Thesis

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 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.

Reference

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.

1 / 1 UNIVERSITE DE GENEVE

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

REMERCIEMENTS

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 !

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 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 ……….…………………..………………………………..….. 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 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

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ABBREVIATIONS

2-DE Two-dimensional polyacrylamide MSA Multiple system atrophy gel electrophoresis NO Nitric oxide α-SYN alpha-synuclein NSAID Non-steroidal anti-inflammatory AC Accession number OGE Offgel electrophoresis AD Alzheimer’s disease OT Orbitrap ALP Autophagy lysosomal pathway MALDI Matrix-assisted laser ATP desorption/ionization BBB Blood brain barrier MPTP 1 - methyl 4-phenyl 1,2,3,6- BG Basal ganglia tetrahydropyridine BP Biological process MS Mass spectrometry CC Cellular component MS/MS Tandem mass spectrometry CID Collision-induced dissociation PAGE Polyacrylamid gel electrophoresis CMA Chaperone-mediated autophagy PANTHER Protein Analysis Through Evolutionnary CNS Central nervous system Relationships CNDP2 Cytosolic non-specific dipeptidase 2 PD Parkinson’s disease CP Central proteome PET Positron emission tomography CSF Cerebrospinal fluid pI Isoelectric point DA Dopamine PMF mass fingerprint DAVID Database for Annotation, PMD Post-mortem delay visualization and integrated discovery PrP Prion protein DBS Deep brain stimulation PTM Post-translational modification DLB Dementia with Lewy bodies RNA Ribonucleic acid ECM Extracellular matrix ROS Reactive oxygen species ESI Electrospray ionization RP Reversed-phase ER Endoplasmic reticulum RR Relative risk FDR False discovery rate SDS Sodium dodecyl sulfate GO ontology SN Substantia nigra pars compacta GWAS Genome-wide association study SNr Substantia nigra pars reticulate GPe/i Globus pallidus internal/external STN Subthalamic nucleus IAA Iodoacetamide TCA Tricarboxylic acid cycle ICAT Isotope coded affinity tags TCEP Tris-2-carboxyethyl-phosphine IEF Isoelectric focusing Ub Ubiquitin IHC Immunohistochemistry UKPDBB United Kingdom Parkinson’s disease brain L-DOPA Levodopa bank LACB Beta-lactoglobulin UPDRS Unified Parkinson’s disease rating scale LB Lewy bodies UPR Unfolded protein response LC Liquid chromatography UPS Ubiquitin proteasome system LCM Laser capture microdissection TOF Time of flight LoC Locus coeruleus TMT(-6) Tandem Mass Tags (sixplex) LPS Lipopolysaccharides WB Western Blot LTQ Linear trap quadrupole

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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, 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 (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.

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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

( (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.

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Chapter I

General introduction

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 Figure 1 Mucuna pruriens. Mucuna pruriens (also known active component of Gastrodia elata herb, as cowage or “Atmagupta” in Sanskrit) is an endemic plant found in India as well as Central and South America could have a neuroprotective activity through containing levodopa. Traditional Indian medicine sources suggest its use to treat PD as far as 5000 BC. 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

11 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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Chapter I : General introduction 13

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.

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

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

13 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- drugs (i.e.alkaloids) and dopaminergic activating agents (i.e. rye based ergot products, ) 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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Chapter I : General introduction 15

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.

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.

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

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Chapter I : General introduction 17

also clinically heterogeneous with different clinical subtypes being recognized based on age of onset, clinical features or progression rate [30].

Motor symptoms Non-motor symptoms Resting tremor: rythmical oscillations of low frequence and variable amplitude, Behavioural and psychiatric problems: expressing involuntarily usually in the hands or feet when the muscles are relaxed depression, anxiety, apathy, hallucinations, fatigue

Bradykineskia/akinesia: slowness of initiation and perfomance (decrease in Cognitive deterioration: mild cognitive amplitude) when performing a volontary or spontaneous movement. Other clinical impairement to dementia (late PD stage) expression: micrographia (progressive smaller handwiriting) , hypomimia (decreased bradyphrenia (slowness of thoughts) facial expression and eye blinking) or freezing (full arrest of movements)

Rigidity: increased muscle tone or resistance felt when by the examiner during the Sensory symptoms : anosmia (loss of smell sense), full range of passive movements. pain (shoulder, back) Postural and gait impairment: Loss of postural reflexes leading to a stooped posture Autonomic dysfunction: orthostatic hypotension, and increasing the fall risk. Slow gait with shuffling steps and decreased arm swings constipation, urinary and sexual dysfunction or festination (fast succession of steps) Others: i.e. dystonia (involuntary intense muscle contractions) Sleep disorders: REM behaviour disorder, daytime sleepiness, restless legs syndrome

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 ). As the disease evolves, disabilities increase with the development of

17 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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Chapter I : General introduction 19

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, -

19 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.

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

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.

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Chapter I : General introduction 21

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, , 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 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. ) 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 inhibitors B (i.e. , ) or DA agonists (i.e. pramipexole). Non-dopaminergic medications comprise several 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

21 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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Chapter I : General introduction 23

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

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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 Figure 7. The Basal ganglia. Coronal functions including movement control and regulation but picture of the brain showing the main basal ganglia nuclei. Image from also cognitive, emotional and motivational processes Neuroscience, fourth edition by D. Purves leading to action.

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Chapter I : General introduction 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.

25

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.

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Chapter I : General introduction 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

27

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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Chapter I : General introduction 29

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.

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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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Chapter I : General introduction 31

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

31 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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Chapter I : General introduction 33

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].

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2.2. Risk factors and etiological hypothesis

2.2.1. Non-genetic risk factors

The cause of sporadic PD is still unknown. Among the many etiological hypotheses, environmental toxin exposure was probably the most studied. The awareness of a relationship with PD was raised during the 1980s, when young individuals developed PD signs after intake of drugs contaminated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a substance similar to the herbicid paraquat [100]. MPTP was then demonstrated to selectively damage the substantia nigra (SN) neurons by blocking mitochondrial complex I [101]. Since then, many pesticides (i.e. rotenone), herbicides (i.e.paraquat) or insecticides were positively associated to

PD risk, with a relative risk (ratio of the probability of having PD in the exposed versus non- exposed group, RR) ranging from 3.7 to 12 [102] potentially in relation with a professional exposure [103]. No convincing evidence implicated metal exposure (i.e manganese, mercury), rural living or well water use as causes of PD [104]. Although many environmental, occupational and life-style risk factors were proposed, older age still remains the most probant and well documented one. Conversely, epidemiological data suggest the existence of protective factors.

The most consistent results were found for cigarette smoking and coffee drinking. A meta- analysis reported pooled RR of 0.39 for current versus never smokers and of 0.69 for coffee drinkers versus non coffee drinkers [105]. The physiological mechanisms of these effects are poorly understood. They might involve neuroprotective action of nicotine through inhibition of

α-SYN fibrillation [106], and through inhibition of adenosine A2 [107]. The use of non-steroidal anti-inflammatory (NSAID) drugs, shown to protect against MPTP neuronal loss in animal models, likely decreases PD risk with a pooled RR for non-aspirin NSAID use of 0.85

[108]. This might indicate a role for inflammation in PD, either causative or consecutive to neurodegeneration. The most recent findings suggest a protective role of high uric acid levels correlated with a slower progression of the disease [109]. Other factors linked to a lower PD risk include intake of vitamin B6, vitamin E (antioxidants) or polyunsaturated fatty acids

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Chapter I : General introduction 35

(neuroprotection, anti-inflammatory), yet without clear evidence [23]. As PD prevalence and incidence are lower in women, sex hormones such as oestrogens have been suggested to exhibit neuroprotective antioxidant properties [18].

2.2.2. Genetic risk factors

2.2.2.1. PD causative genes

Monogenetic causes may not assume a predominant role in PD, as a clear mendelian inheritance can only be established in about 10% of the patients. A study including 193 men twins with PD reported a substantial influence of genetic over environmental factors for the 16 young onset cases, with concordance rates of 100% in monozygotic and 12% in dizygotic twins

[110]. However, overall concordance rates did not differ by zygocity, indicating little importance of genetic component at the population level when the disease begins after the age of 50. The first mutation was identified in SNCA - the gene encoding α-SYN - in 1997 [111], with additional point mutations, duplications and triplications identified in familial PD kindred with autosomal dominant inheritance [112-114]. Interestingly, α-SYN protein turned out to be a major component of LB [115] and SNCA duplications were recently associated to PD sporadic cases

[116]. Since then, at least 10 other causative genes have been associated to autosomal dominant

(i.e SNCA, UCHL-1, LRRK2) or autosomal recessive (i.e Parkin, PINK1, DJ-1) PD and have been extensively reviewed [117]. A list of the causal and susceptibility genes known up to 2009 with details on mutations, mode of inheritance, gene function or clinical features is provided in Chapter 2, Table 2. Since then, two new autosomal dominant genes, VPS35 (PARK17) [118] and EIF4F1 (PARK18) [119] were found in kindreds presenting late-onset PD. However, familial forms of PD often display atypical clinical symptoms - such as young onset or dystonias - and histological features such as absence of LB, constituting a particular category of PD cases.

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2.2.2.2. Susceptibility genes

Most PD cases are sporadic, likely caused by a complex interaction between genetics and environment rather than highly penetrant genes. Specific genetic polymorphisms, may be associated to PD susceptibility although they cannot explain on their own the disease status.

Association studies allow the identification of those variants, that compared to highly penetrant mutations are more common in the population (about 1%) and have a limited effect on the disease phenotype. To date, more than 800 genetic association studies in PD have been published [120] to decipher the missing heritability in PD, with a continual update provided on the PD gene online database [120]. However, inconsistent results were often found between such studies that usually lacked power with too limited sample sizes. Candidate gene studies assessed the contribution of a few “PARK” genes -responsible for monogenic PD forms - as well as selected genes involved in other neurodegenerative diseases (i.e. Alzheimer’s disease,

Gaucher’s disease) to the risk of developing idiopathic PD. A few of them such as SNCA (α-

SYNuclein, PD)[121], LRRK2 (PD)[122], MAPT (microtubule associated protein tau, AD) [123] or

GBA (glucocerebrosidase, Gaucher’s disease) [124] gene loci significantly appeared to impact PD susceptibility. Recently, meta-analyses were performed using most of PD gene website resources, either GWAS or smaller scale studies, that showed genome-wide statistically very significant association of eleven loci BST1, CCDC62/HIP1R, DGKQ/GAK, GBA, LRRK2, MAPT,

MCCC1/LAMP3, PARK16, SNCA, STK39, and SYT11/RAB25 and novel evidence for ITGA8 polymporphism [120]. However, methodology must be improved to identify the entire genetic component underlying sporadic PD, as only 60% of the population-attributable risk might be explained by the most promising PD loci identified until now [125].

To conclude, although single environmental toxins (i.e. MPTP) or single gene mutations

(i.e SNCA, Parkin) have been shown to cause rare PD forms, idiopathic PD probably results from a complex combination of environmental factors, genetic susceptibility and age. The understanding of the mechanisms underlying the main PD risks and protective factors may help

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Chapter I : General introduction 37

to delineate the molecular events at work and lead to the development of neuroprotective treatment. For example, genes identified in familial PD or suggested to predispose to PD fall in several functional categories that might also been perturbed in sporadic PD. Some of them code for proteins involved in mitochondrial function (i.e. PINK-1, DJ-1, Omi/HtrA2), polypeptide degradation pathways either through ubiquitin proteasome system (UPS) (i.e. parkin- DJ-1,

UCHL1) or lysosomes (i.e. GBA) as well as protein/organelle trafficking and vesicular function (i.e. a-SYN, tau, vps35). Other genes including LRRK2 are still poorly characterized and a better understanding of their cellular role might lead to novel insights in PD etiology and pathogenesis.

2.3. Pathogenic mechanisms of PD

Despite the clues provided by genetic breakthroughs and the many alterations observed in the brain of idiopathic PD cases, the molecular mechanisms underlying sporadic PD pathogenesis and particularly the massive and selective neurodegeneration in the SN still need to be deciphered. It is generally thought that a combination of environmental factors along with aging process initiate a cascade of pathological cellular and molecular events in genetically susceptible individuals, ultimately leading to neuronal demise. Many mechanisms have been shown to sensitize neurons to death and induce protein aggregation, including protein degradation system impairment, mitochondrial dysfunction and oxidative stress, inflammation, excitotoxicity or apoptosis. In all likelihood, more than one of the proposed hypotheses might be at work in PD but the exact combination and succession of events still need to be established. In addition, the specific morphological and physiological characteristics of the affected neuronal populations might account for their particular vulnerability. In the next paragraphs, the particular features of SN DA neurons that might predispose them to cellular stress will be reminded. Then, the main pathogenic mechanisms will be briefly discussed, with a special emphasis on inflammation and lysosome/mitophagy hypotheses. As those issues are already reviewed in Chapter II, some details will be omitted.

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2.3.1. The specific vulnerability of SN DA neurons

Considerable knowledge has been gained in the last decades on pan-cellular etiological factors negatively or positively impacting non-neuronal and neuronal cell types in PD. The selective vulnerability of nigral dopaminergic neurons, which represent less than 0.0001% of all brain neurons, could be attributed to cell-specific risk factors (see review in Chapter II, “3-

Selective degeneration”). Briefly, DA has been seen as a culprit, because its metabolism was shown to generate toxic reactive oxygen species (ROS)[126]. However, a variety of non-DA neurons also die in PD and conversely some DA neuron populations are spared (see 2.1.1.3

“Beyond SN”), arguing against DA as the principal cell-risk factor. SN DA neurons, as well as other neurons damaged in PD, have a distinctive impressive axonal field with disproportionally long unmyelinated axonal projections, each of them supporting no less than 370’000 synapses

[127]. Comparatively, SN DA cell body is small, representing about 1% of the total cell volume

[126]. The size and complexity of these neurons imply an elevated axonal trafficking and a high

ATP demand, which might sensitize them to proteostatic stress, aggregation and energetic crisis.

This could explain why mutations in genes related to mitochondrial and trafficking activities could predispose to PD. Moreover, adult SN DA neurons have a particular and uncommon physiological phenotype. They are neuronal pacemarkers, exhibiting an autonomous activity in the absence of synaptic input to help maintaining DA levels in the striatum, the main projection region. For that, they rely on relatively rare L-type Ca2+ channels Cav1.3, which induce broader action potentials. Contrasting with what occurs in the majority of neurons, those channels are opened frequently with larger magnitude of Ca2+ influx [128]. The resulting Ca2+ overload could underlie cellular stress and be responsible for SN DA neuron specific vulnerability. Any impairment in Ca2+ homeostasis regulation mechanisms such as ATP-dependent pumping as well as mitochondrial and endoplasmic reticulum adequate buffering function might critically compromise SN DA neurons survival. These neurons might additionally exhibit a lower intracellular Ca2+ buffering capacity sensitizing them to Ca2+induced stress. They are typically

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located in regions where calbindin, a Ca2+ fast buffering protein, is poorly expressed, whereas neurons in calbindin rich regions are relatively more resistant to death in PD [77, 78]. Finally, glutamatergic input to SN could also sensitize them to death through excitotoxicity mediated by

NMDA receptor activation [129]. The specific factors characterizing SN DA neuron phenotype are important as they provide clues to explain the pattern of neuronal loss and their relative susceptibility to the molecular pathogenic mechanisms such as mitochondrial impairment that will be described below. They could as well lead to the establishment of new therapies, such as the administration of L-type Ca2+ channels antagonists [129].

2.3.2. Potential mechanisms underlying neurodegeneration in PD

2.3.2.1. Alpha-synuclein, Lewy bodies and protein aggregation

The deposition in brain tissues of insoluble aggregates containing abnormal proteins is the pathological hallmark of many neurodegenerative disorders including PD. In these conditions, accumulation of misfolded, abnormally modified (i.e. by post-translational modifications, mutations) or proteotypically cleaved proteins may ultimately lead to the formation of insoluble fibrillar deposits such as typical LB in PD. Alpha-SYN seems to play a central role in PD, although its mechanisms of aggregation and toxicity remain unraveled.

Pathogenic mutations and multiplication of SNCA gene, respectively inducing α-SYN misfolding and wild-type α-SYN overexpression, were both demonstrated to promote α-SYN aggregation and neurodegeneration in familial PD cases. Remarkably, aggregated α-SYN has then been identified as a major constituent of LB in sporadic PD. Post-translational modifications of α-SYN

(nitration, phosphorylation, glycation, glycosylation ubiquitination, dopamine adducts), or truncated forms of the protein observed in the LB of sporadic cases and/or PD models, were shown to lead to toxic protein properties and eventually a higher propensity to aggregation

[130]. It is still unclear if LBs represent a neurotoxic insult or if they are formed as a defensive measure to tackle misfolded or modified proteins, similarly to aggresomes. In fact, α-SYN

39 aberrant soluble oligomeric conformations also known as protofibrils might be the more toxic entities. They were demonstrated to mediate neuronal death through various targets and effects, including proteasome inhibition [131] or disruption of mitochondrial and vesicular membranes [132]. Interestingly, α-SYN may propagate from a neuron to another following a prion-like process and account for the disease propagation throughout the CNS [94]. Recently, the genetic association between SNCA and sporadic PD has been confirmed in a large GWAS study.

The involvement of protein aggregation in PD pathological process is undoubtless, either causing or being the consequence of pathological alterations in susceptible neuronal population.

Increasing numbers of proteins are being identified in LB, indicating that α-SYN might not be the only key player. Other LB aggregation-prone constituent such as parkin might be pathogenic in

PD as well. Unraveling the exact composition of LB could provide some clues on other potential proteins playing a role in PD neurotoxicity. Under pathological conditions such as proteostatic impairment or during normal aging, the propensity to protein misfolding and aggregation might be enhanced.

2.3.2.2 Impairment of protein degradation system

Molecular chaperones, ubiquitin-proteasome system (UPS) and autophagy-lysosomal pathway (ALP) are the main cellular pathways promoting the folding or the degradation of unwanted proteins. Whereas UPS degrades mostly short-lived soluble or misfolded proteins, ALP refers to the lysosomal degradation of intracellular components including proteins and organelles. Dysfunction in any of these pathways may lead to protein accumulation and aggregation resulting in cellular toxicity. These mechanisms have been consistently implicated in

PD pathogenesis, either as a cause or a consequence of the neurodegenerative process in at risk cell populations. Both UPS and ALS might contribute to α-SYN turnover and pathology [130].

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Age, the main PD risk factor, might predispose to impairment in protein degradation system, as both UPS and ALP activities are found to be decreased in old individuals.

2.3.2.2.1. Ubiquitin proteasome system

The UPS is the major pathway that mediates the selective degradation of intracellular and membrane proteins as well as misfolded proteins in the cytosol, nucleus and ER. Targeted proteins are tagged with ubiquitin (Ub) chains, the proteasomal recognition signal, through the sequential action of several enzymes - Ub-activating enzymes (E1), Ub-conjugating enzymes (E2) and Ub (E3). The large ATP-dependent 26S proteasome consists of two capping 19S regulatory subunits for recognition and a catalytically active 20S core degrading proteins into peptide fragments that will further be processed by peptidases. Ub is recycled by Ub , which Figure 13. Schematic representation of the disassemble polyUb chains into monomeric Ub ubiquitin proteasome system (UPS). Image adapted from Rubinsztein DC et al., Nature, 2006. (Figure 13).

The identification of mutations in Parkin, an E3 , and ubiquitin C-terminal hydrolase

L1 (UCHL-1), a de-ubiquitinating in monogenic PD forms, suggested a link between PD pathogenesis and UPS. LB were found to contain highly ubiquitinated proteins [133] as well as

UPS components including proteasomal subunits as well as ubiquitinating and de-ubiquitinating enzymes [134-136]. The first evidence of UPS impairement in sporadic PD came from the

41 observation of decreased proteasomal activity in post-mortem brain tissue of PD cases compared to controls, specifically in the SN [137, 138]. In addition, some studies reported reduced proteasome activity and expression of UPS components in peripheral blood cells of PD patients [139]. Proteasome inhibitors were shown to be sufficient to induce protein aggregation and neurodegeneration in vitro and in vivo [140-142]. Exposure to toxins (MPTP, rotenone) or to high levels of α-SYN was shown to alter proteasome function and trigger neurodegeneration in

PD models [142-144]. The reciprocal effects of proteasome dysfunction and α-SYN overexpression might indicate a self-perpetuating process in neuronal tissues: increased α-SYN levels alter UPS function, in turn leading to impaired α-SYN clearance and formation of α-SYN- containing inclusions [142]. As UPS is an ATP dependent process, any defect in energy metabolic pathways could impair its function.

2.3.2.2.2. Lysosomes & chaperone mediated autophagy

ALP comprises three distinct autophagic pathways, differing in the way substrates are delivered to lysosomal lumen: macroautophagy (generally referred to as autophagy), chaperone- mediated autophagy (CMA) and microautophagy (Figure 14). Lysosomes are vesicles, which degrade various cellular components including proteins and organelles through the action of hydrolases at an acidic pH maintained through ATP-dependent proton pumps. Contrasting with

UPS, macroautophagy is primarly involved in the degradation of long-lived stable intracellular proteins, protein aggregates or organelles such as mitochondria, and can be induced under cellular stress such as starvation [145]. Of note, large proteins or protein aggregates unable to pass through the proteasome barrel can be eliminated by autophagy [146, 147]. Autophagy is characterized by a multi-step process involving the “bulk” degradation of entire cytosolic regions, through the formation of double-membrane vesicles termed autophagosomes. Later, these autophagosomes fuse with lysosomes or late endosome, providing the necessary proteolytic enzymes for degradation. The fusion product or autolysosome, is finally dismantled

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while its content is digested. In CMA, specific cytosolic proteins are recognized by molecular chaperones (i.e. Hsp70) through a particular amino acid motif and degraded by lysosomes after their internalization mediated by Lamp-2a receptor. Microautophagy, the less characterized autophagy subtype, refers to the process by which lysosomes directly sequester cytosolic components at their surface, by invagination of the lysosomal membrane.

Figure 14. Schematic representation of the cellular autophagy lysosomal pathway. Three different types of autophagy have been described: macroautophagy, chaperone-mediated autophagy (CMA) and microautophagy. In macroautophagy, intracellular components ( i.e proteins, mitochondria) are sequestered by a limiting double membrane to form an autophagic vacuole further fusing with lysosomes. In CMA, selective substrate proteins are targeted to lysosomes through lysosomal receptor (LAMP-2A) binding. In microautophagy, substrates are internalized through lysosomal membrane invaginations. Image adapted from Martinez-Vicente et al Lancet neurology, 2007

ALP is a critical cellular process involved in the clearance of misfolded proteins or protein aggregates. Dysfunction of the ALP can occur at many steps and ultimately lead to the accumulation and aggregation of unwanted proteins leading to cell death [146, 147]. The involvement of ALP in PD neurodegenerative process has been supported by several findings.

The identification of a mutation in a lysosomal ATPase (ATP13A2) in juvenile onset form of PD

[148] as well as the GWAS association of lysosomal glucocerebrosidase (GBA) to sporadic PD, have suggested that lysosomal alteration could lead to α-SYN aggregation and neurodegeneration. Mutations in LRRK2, responsible for autosomal-dominant PD, might also significantly impair the autophagic pathway [149, 150]. Interestingly, autophagosomes and other

43 autophagic structures were found to accumulate in post-mortem SN tissue of PD patients [151].

Further studies indicated an increase in the autophagosome marker LC3-II [152, 153] and a decrease of the lysosomal marker LAMP-1 [154, 155] in PD, supporting the existence of abnormal autophagy in PD pathology [142]. It is still unclear whether autophagy is a causative or a protective factor of neurodegeneration. Depending on the point of view, the increased number of autophagic vacuoles observed in PD patients might result from an aberrant activation of autophagy possibly inducing neurodegeneration. Alternatively, they might represent a cytoprotective response to enhance the degradation of abnormal proteins, possibly following

UPS impairment. Indeed UPS inhibition was found to upregulate autophagy and might constitute the default degradation pathway [156]. The interplay with α-SYN has also been investigated. α-

SYN might be cleared by both UPS and autophagy pathways [157]. CMA was shown to selectively mediate native α-SYN degradation while failing to clear misfolded α-SYN form [158]. As a result,

α-SYN mutant might accumulate and even inhibit CMA [158], inducing a perturbation in proteostasis and contributing to neurotoxicity [158]. The autophagic pathway has also been shown to participate in mitochondria turnover through a specific form of macroautophagy called mitophagy. Genetic findings support a role for impaired mitophagy in PD pathogenesis. In autosomal recessive PD forms, mutations were found in Parkin and PINK-1 [159, 160], two proteins important for mitochondrial maintenance and likely involved in signaling pathways controlling mitophagy [161].

In PD pathology, insufficient autophagy activation might occur and impair the degradation of protein aggregates and defective mitochondria, which might in turn induce its inhibition. The role of excessive activation of autophagy remains unknown and its elucidation might be a prerequisite for the development of therapeutic strategies eventually enhancing autophagy (i.e. mTor inhibitors).

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2.3.2.3. Mitochondria and oxidative stress

Strong evidence supports a role for mitochondrial dysfunction and oxidative stress in PD pathological processes. Mitochondria are highly dynamic organelles assuming a variety of essential cellular roles including energy production by oxidative phosphorylation - whereby they are the major source or reactive oxygen species (ROS), regulation of Ca2+ homeostasis and programmed cell death (apoptosis). Any functional alteration in mitochondria might impact many processes and thus be critical for cell demise, as schematized in Figure 15. Given their particular biochemical, morphological and physiological characteristics, neurons rely on high rates of metabolic activity and energetic demand and might be particularly sensitive to mitochondrial alterations.

Figure 15. Potential pathological cellular effects of mitochondrial dysfunction in PD. Toxins, misfolded proteins or oxidative stress can all impair mitochondrial function, in turn altering various cellular processes (red) leading to cell demise. Of note, all ATP-dependant mechanisms such as UPS can indirectly be altered by the consecutive energy depletion. AIF, apoptosis-initiating factor; UPS, ubiquitin–proteasomal system. Image from Winklhofer , K et al., Biochimica et Biophysica Acta (BBA), 2010.

The first links between PD and mitochondria came from the observation of complex I activity deficiency in MPTP induced parkinsonism [100] and in the SN of sporadic PD patients at autopsy [162]. Inhibition of complex I activity by a variety of mitochondrial toxins (i.e. MPTP,

45 rotenone) was shown to induce PD features in both human and animal models of PD [163-167], and might be mediated by α-SYN [168]. Reduced complex I activity was also recently found in the platelets of PD patients, indicating an eventual systemic complex I defect in about 25% PD patients [169]. Deficiency in electron transport chain such as complex I inhibition, may decrease

ATP production as well as increase ROS formation and oxidative stress levels. A vicious cycle might then be installed, whereby mitochondria are in turn a source and a target of ROS, ultimately leading to the loss of vulnerable cell populations such as SN DA neurons. Oxidative damage has consistently been observed in the SN of sporadic PD brains [170] and LB were shown to contain abnormal oxidized forms of α-SYN (i.e. nitrated) [171]. Other sources of ROS include DA metabolism, reactive iron deposition and impaired antioxidant pathway.

Recent findings suggest that mutations in the mitochondrial genome (mtDNA) are involved in PD pathogenesis. MtDNA together with nuclear DNA, encodes proteins from the respiratory chain. Altered oxidative phosophorylation was observed in animal models exhibiting defective mtDNA (reviewed in [172]). A deletion in TFAM, a mitochondrial transcription factor, in

DA neurons was shown to reproduce PD features such as a progressive DA neurodegeneration in a mice model [173]. In the SN neurons of PD patients, mtDNA deletions were found that appear to increase along with age [174]. Substantial insights in the understanding of mitochondrial role in PD came from the identification of inherited mutations in genes linked to mitochondria and oxidative damage, including: PINK1 (mitochondrial kinase), DJ1 (antioxidant), Parkin (ubiquitin

E3 ligase), ATPA13A2 (lysosomal ATPas), LRRRK2, SNCA, omi/Htra2 (mitochondrial kinase) [175,

176]. Moreover, GWAS identified genetic risk factors associated to sporadic PD which strikingly overlapped mendelian genes (LRRK2, SNCA), indicating the familial and sporadic PD might share common pathways. The characterization of these genes has helped to dissect the diverse aspects of mitochondrial activity affected in PD, including impairment in mitochondrial biogenesis, fission/fusion balance, mitophagy or mitochondrial stress for instance (Figure 16).

Altogether, strong evidence has been provided for the occurrence of mitochondria defects and

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oxidative stress in PD and therapeutic strategies targeting some of these processes may hold great promise.

Figure 16. PD-associated genes affecting mitochondrial function and oxidative stress. Rare inherited mutations in genes involved in mitochondrial function such as Parkin, α-SYN, PINK1, DJ-1, LRRK2, HTRA2, but also ATP13A2 (not represented here) all lead to PD variants. ATP13A2 codes for a lysosomal ATPase and was recently shown to regulate mitochondrial biogenesis through mitophagy. HTRA2, high temperature requirement protein A2; LRRK2, leucine-rich repeat kinase 2; PD, Parkinson disease; PINK1, PTEN-induced putative kinase 1. Image from Henchcliffe C. et al, Nature Clinical Practice Neurology, 2008.

2.3.2.4. Glial reaction and inflammation

Substantial involvement of glial cells in the initiation and progression of DA neuron demise has been postulated with the findings of inflammatory features in parkinsonian brains

(Figure 17). A strong glial reaction along with an increase in the expression of proinflammatory factors (i.e tumour necrosis factor (TNF)-α, interleukin (IL)-1β, interferon (IFN)-γ) [177, 178]) or enzymes associated with inflammation (inducible nitric oxide synthase iNOS or cyclooxygenase

47

COX2 [179]) were observed at the time of death in the SN of PD patients, which might contribute to the neurodegenerative process [180-182]. Much evidence points to a particular vulnerability of dopaminergic cells to inflammatory attacks in PD. Epidemiological studies have suggested that non-steroidal anti-inflammatory drugs (NSAIDs) could have a protective effect against neurodegeneration as regular consumers are relatively spared by PD [183, 184]. In vitro exposure of various neuron-glial cell cultures to lipopolysaccharides (LPS) endotoxin known to activate glial cells and cytokine expression, was shown to induce dopaminergic neurodegeneration [185, 186]. A single intranigral injection of LPS in living rats seems to selectively damage SN while preserving other neuronal types (serotoninergic, GABAergic) and anti-inflammatory intervention with dexamethasone can attenuate LPS toxic effect [187]. All these aspects imply that inflammation processes may mediate DA neuronal loss.

Different subpopulations of glial cells (microglia, astrocytes, oligodendrocytes) may contribute actively to DA neuronal demise [188]. The function of astrocytes - the more abundant glial cells - in inflammation is not clear. They appear to become reactive and able to wall off pathological targets, probably stimulating microglia while releasing protective compounds to the surroundings [183]. They are known to secrete pro-inflammatory agents such as ICAM-1 observed in the SN of PD [189] and whose secretion could be activated by monomeric forms of

α-SYN itself [190]. Astrocytes may play a dual role in PD as they have also been shown to secrete anti-inflammatory agents (ie glial cell-line derived neurotrophic factor (GDNF) or brain derived neurotrophic factor (BDNF) [191, 192] as well as factors inducing antioxidant enzymes such as

NRf-2 whose overexpression was reported to protect against 6-OHDA damage in mice [193].

Very little is known about oligodendrocytes role in PD.

The most interesting cells seem to be the microglia or the immune competent cells of the CNS. Through the monitoring of the area they reside in, they can detect immune insults, pathogens or change in their microenvironment [194]. They secrete a multitude of immunomodulatory molecules (pro-inflammatory or anti-inflammatory cytokines,

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neurotrophins, reactive oxygen species, nitrogen species etc), which communicate signals to surrounding cells and can phagocyte pathological compounds. Microglia becomes active through the binding of extracellular stimuli to their surface receptors [194]. For example, cytokines produced by microglia [195], misfolded or aberrant proteins such as aggregated α-SYN [196],

ATP released by dying neurons or matrix metalloproteinase-2 involved in neuromelanin degradation can all activate microglia. Whereas microglia activation is beneficial to normal brain function, it can be deleterious when over- or continuously activated by endogenous proteins, toxins and pathogens, or via damaged neuronal signals [194]. Microglial-derived free radicals can cause neuronal death [197] and could be implicated in PD pathogenesis. An acute insult can initiate a self-sustaining inflammatory reaction maintained by a positive feedback from dying neurons [183]. This vicious cycle amplifying neurons destruction and referred to as reactive microgliosis is characteristic of neurodegenerative diseases [198].

Glial dysfunction seems to participate actively in the PD SN pathology through inflammatory mechanisms that are still elusive. In a first scenario, inflammation process may be a secondary event consecutive to neurodegeneration in PD which could aggravate the pathologic process and account for the continuous worsening of the disease [187]. Alternatively, inflammation could be directly responsible for neurodegeneration or increase neuronal vulnerability to neurotoxins through the release of toxic substances. Interestingly, aggregated proteins such as α-SYN [196] can induce neuronal death through microglial activation. In a therapeutic point of view, neuro-inflammation blockage could represent a valuable strategy.

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Figure 17. Inflammation in Parkinson's disease. Aggregates or oligomers of α-SYN released by affected neurons may activate microglia, which in turn induces the production of ROS and the expression of proinflammatory mediators through NF-κB induction (inhibited by Nurr1). These factors may directly act on DA nigral neurons and activate astrocytes. Molecules secreted by microglia and astrocytes might together promote neuronal demise through apoptosis and necrosis, amplifying the inflammatory process. Microglia can also play a protective role by mediating the clearance of protein aggregates. Image from Glass CK et al., Cell, 2010.

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3. PROTEOMICS AND PARKINSON’S DISEASE RESEARCH

3.1. “omics” and PD research

Despite intensive research, the etiopathological mechanisms underlying sporadic PD remain unknown, impeding the development of disease-modifying strategies able to stop or slow the disease progression. 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 PD. The recent emergence of new “candidate-free” unbiased disciplines such as proteomics but also genomics and GWAS (already discussed in PD susceptibility risk factor, 2.1.2.2), transcriptomics, or metabolomics has boost the exploration of new avenues to decipher molecular pathways at the basis of PD and may help to define PD biomarkers. Whereas proteomics focuses on the characterization of changes related to proteins in a sample (see next section “Proteomics”), transcriptomics refers to the identification and measurement of all transcripts (mRNAs, non- coding RNAs and small RNAs). Metabolomics concerns the comprehensive monitoring of metabolites – low molecular weight entities chemically processed during cellular metabolism - in biological systems. These strategies can generally be applied to various samples, including biofluids (blood, serum, plasma, CSF, urine…) and tissues for the comparison of their "disease” versus “healthy” states. Given the current limitations of animal models (discussed in Chapter III), this section will cover human sample-based analyses only.

Transcriptome profiling has generally been achieved by hybridization-based cDNA microarrays, progressively replaced by the recently developed RNAseq deep-sequencing technology [199]. Several transcriptomics studies have been published investigating SN autopsy tissue [200-202] and laser-capture microdissected (LCM) DA neurons ([203, 204] or biological fluids (i.e. plasma [205]) in PD individuals. A common tendency was observed towards a dysregulation in PARK genes as well as genes involved in protein degradation (UPS, ALP), energy metabolic pathways (i.e. glycolysis), synaptic transmission, oxidative stress, inflammation and

51 apoptotic cell death. The apparent lack of concordance observed between transcriptomics studies could be due to the utilization of gene lists for comparison rather than standardized pathways, which indicated a common decreased expression of DA receptor and insulin growth factor 1 (IGF1) signaling in PD [206]. A recent study using a system biology approach to integrate both PD GWAS and gene expression data identified consensus disease pathways in PD including axonal guidance, Ca2+ signaling or focal adhesion [207].

Metabolic profiles act as molecular signatures for biochemical activities characteristic of a physiological or disease state [208]. The most popular analytical approaches in metabolomics are either nuclear magnetic resonance (NMR) or LC-MS and GC-MS, which enable the rapid profiling of thousands of metabolites simultaneously (reviewed in [208]). The few studies investigating disrupted metabolic profiles have provided evidence for a role of uric acid, a natural antioxidant, in PD development. Reduced urate levels were found in serum, CSF and in

SN, correlating with DA neurodegeneration, advanced PD symptoms and higher risk for developing PD [209-211]. In addition, increased levels of other oxidative markers (i.e. glutathione) were found in the plasma of PD patients [212]. Recently, a group managed to distinguish different plasma metabolic signatures between sporadic PD cases, PD patients carrying G2019SLRRK2 mutations, asymptomatic G2019SLRRK2 carriers and controls including decreased urate levels in PD patients [213]. Altogether, these data provided further insights into the oxidative stress pathological hypothesis of PD. Within metabolomics, lipidomics is an emerging field allowing the characterization of the enormous variety of (i.e. fatty acyls, glycerolipids, glycerophospholipids, sterol lipids and sphingolipids) and interacting partners at a biological system level [214]. levels are tightly regulated to assume crucial structural, metabolic or regulatory roles in cells or tissues. Alterations in lipid homeostasis have been implicated in PD, where it may induce neurodegeneration. Lipids are known to be associated to

LB [215] and α-SYN is a lipid binding protein that could associate with oxidized metabolites, resulting in mitochondrial dysfunction and dopaminergic neuron demise [216]. Recently, the first

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lipidomics analysis of human PD disease tissues was published, indicating significant changes in lipid biosynthetic pathways (i.e. oxysterol increase) in the visual cortex (VC) of PD patients in the absence of obvious pathology [217]. These changes might be neurotoxic and account for the visual hallucinations experienced by PD patients [217]. The current limited number of lipidomics studies reflects the complex nature of lipids and the limitations of the analytical tools available.

Finally, proteomic tools, which appear to be particularly promising and well-suited to capture the complex nature of PD, will be discussed in the next section. Overall, the integration of all “omics” disciplines through system biology approaches will contribute to provide a more comprehensive picture of the pathological processes leading to neurodegeneration in PD (Figure

18).

Figure 18. The “omics”cascade. The ‘omics’ cascade integrates the flow of biologic information at a system level, from DNA (genomics), transcribed to mRNA (transcriptomics), translated into protein (proteomics) which catalyze reactions giving rise to metabolites (metabolomics) as well as glycoproteins and carbohydrates (glycomics), and lipids (lipidomics). Modified from Di Leo et al, Ann Onc, 2007

3.2. Proteomics

3.2.1. Generalities

Proteomics is a particularly prominent “omics” discipline providing a unique window into complex molecular regulatory networks through the systematic characterization of entire sets of

53 proteins expressed in a cell or a tissue at a given time [218]. The direct study of proteins might be regarded as an inescapable source of knowledge as (i) proteins are the principal effectors in biologic systems and (ii) as neither the genome nor the transcriptome can reflect the proteome complexity. A genome of 20’000 genes produces about 150’000 transcripts and 1’000’000 proteins, as a result of alternative splice variants, RNA editing or PTMs together establishing the rich variety of protein isoforms. Moreover, mRNA levels do often not correlate with protein levels and activity [219]. The proteome is thought to be highly dynamic with protein concentrations separated by up to 10 orders of magnitude in serum for example [220], and can vary according to protein stability and turnover, localization (tissue, cell-type, compartment) or temporal expression. Because of its size, dynamics and unknown complexity, the complete analysis of a proteome represents a considerable challenge, which is still not achievable using a single method.

Large-scale proteomic analyses typically require four consecutive steps: sample preparation, protein/peptide separation, mass spectrometry (MS) instrumentation and finally bioinformatics data processing. Two fundamental MS-based proteomic strategies can be distinguished. In “Top down” approach (not covered in this section, see review in [221]), intact proteins are analyzed by MS. In “Bottom up” approach, proteins are proteolytically cleaved into by site-specific enzymes such as trypsin prior MS analysis. A wide range of proteomic workflows is available either “gel-based” - relying on protein separation by one (1-DE) or two dimensional polyacrylamide gel electrophoresis (2-DE), or “gel-free” also known as “shotgun approach” - involving protein/peptide fractionation by multidimensional liquid chromatography

(LC) or Offgel (OGE) combined to label-free, SILAC, ICAT or TMT/iTRAQ for protein quantification.

These proteomic approaches have allowed the identification of thousands of proteins from complex mixtures together with the determination of their relative abundance, post- translational modifications (PTMs), protein-protein interactions or structure. In PD, the large amount of proteomic data generated has greatly contributed to the investigation of disease

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molecular pathways and biomarkers, a subject extensively covered in the review presented in

Chapter II. Consequently, this chapter will rather focus on proteomic basic tools and workflows applied to PD research.

3.2.2. Sample preparation

In PD, most studies have relied on the proteome analysis of cerebrospinal fluid (CSF) and brain autopsy tissues. CSF may represent an ideal source of diagnostic biomarker, potentially reflecting the disease pathology due to its proximity to brain structures undergoing neurodegeneration. CSF can be easily obtained by lumbar puncture during patients’ life. Brain autopsy tissues, in particular the SN region, have been widely used to gain new insights in PD pathogenesis, allowing the targeting of the primarily affected structures. We recently reappraised their validity (see Chapter II). One of their principal advantages is the post-mortem confirmation of PD diagnosis, as PD still cannot be diagnosed accurately within patient’s life time.

Adequate sample collection, handling and storage are crucial to prevent analytical bias and allow reproducible comparisons between samples [222]. For example, blood contamination in CSF samples leaded to conflicting results when measuring α-SYN levels in PD patients for biomarker research purpose [57]. Temperature was also shown to affect protein integrity.

Consequently, when studying autopsy tissues, a particular care must be taken to minimize post- mortem delay (PMD) - the time elapsed between death and sample processing or freezing at -

80°C, ideally under 48 hours, at which most changes might occur at room temperature [223].

Efforts are generally also placed into sample sub-fractionation at a tissular, cellular or subcellular levels to reduce sample complexity and target the most relevant proteomes. CSF and blood are typically depleted of their few highest abundant proteins using immunoaffinity columns (i.e.

MARS column) to enrich in the many lower abundant proteins that could be potential markers of pathological states [224]. When using PD autopsy samples, increasing levels of specificity can be

55 assessed with sub-proteome analyses of entire cryo-dissected brain regions such as the cortex

[225] or the SN [226-228] down to various sub-cellular fractions of interest such as mitochondria

[229], synaptosomes [225] , cortical LBs [230, 231] or neuromelanin granules [232].

3.2.3. Sample separation

An effective fractionation is critical prior to MS in order to increase the detection coverage, as peptide mixtures generated by the digestion of complex samples generally exceed the resolutive power of mass spectrometers. A variety of separative methods has been developed to reduce complexity at the protein and/or peptide level, either by electrophoresis

(i.e. SDS-PAGE, IEF, Offgel, CZE), chromatography (i.e. SCX, RP) or immunoaffinity.

Multidimensional fractionation can be implemented to enhance proteome coverage and detection sensitivity in MS.

Two-dimensional gel electrophoresis (2DE), which combines isoelectric focusing (IEF) in the first dimension and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second, is commonly used to efficiently separate complex protein samples [233]. IEF separates proteins according to their intrinsic charge or isoelectric points (pI). Proteins, which are amphoteric molecules carrying both positive and negative charges, migrate in a pH gradient upon electrical field application and focus around the pH at which their net charge is null (=pI). In

2-DE, immobilized pH gradient (IPG) strips (i.e. pH 3 to 10) are used, in which the gradient molecules are pre-cast in gel. SDS-PAGE separates proteins according to their electrophoretic mobililty, correlating with their molecular weight. Proteins are typically solubilized by SDS denaturation, heat, disulfide bond reduction (i.e. TCEP) and alkylation (i.e. IAA), to prevent disulfide bond reformation (i.e with iodoacetamide) before being loaded on gel. After migration in polyacrylamide matrix upon electrical field application, proteins can be detected by different stains varying in their detection range. Coomassie Brilliant Blue is the less sensitive, followed by

Sypro Ruby, which uses fluorescent dyes, and silver staining being the most sensitive [234]. MS-

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based identification can be performed after in-gel tryptic digestion. 2-DE can resolve routinely about 2000 proteins simultaneously, reflecting changes in protein expression levels, isoforms or

PTMs, with detection range down to 1 nanogram per spot. Major drawbacks include i) under- representation of membrane, highly acidic or basic, and very small or large proteins ii) co- migration of proteins leading to multiple protein per spot iii) time-consuming identification without high throughput automation iv) gel-to gel variations hampering comparisons [235].

Some of the reproducibility issues can be overcome by difference gel electrophoresis (DiGE) technology, in which up to three samples can be simultaneously analyzed in a single gel using different fluorescent dyes [236]. Alternatively to conventional IEF procedure, a recently developed technique termed Off-gel (OGE) allows the collection of peptides or proteins samples in liquid phase after IEF [237, 238]. OGE fractions can be submitted directly to MS without the need for any gel extraction procedure, or further separated by gel-based (i.e. 1-DE, 2-DE) or liquid chromatography (LC) techniques for instance.

Liquid chromatography (LC)-based techniques are commonly used in shotgun proteomic approaches, more suitable for the rapid identification and quantification of proteins in complex mixtures. Proteins or peptides can be separated based on their physicochemical properties such as hydrophobicity (i.e. C4, C8, C18 columns), ionic charge (i.e. Strong Cation Exchange SCX), size, or affinity using specific antibodies (i.e. MARS column) or metal binding (i.e. immobilized metal affinity chromatography IMAC). Typically, reversed-phase (RP) columns allow the separation of proteins or peptides according to their degree of hydrophobicity. Their stationary phase generally consists in carbon chains of variable length, C4 or C8 for proteins and C18 for peptides.

Elution with a volatile MS-compatible organic solvent mobile phase (i.e. acetonitrile) allows LC to be directly coupled with MS allowing for automated high throughput analyses. These chromatographic techniques can also isolate informative subsets of proteins or peptides carrying phosphorylation, glycation, glycosylation or being cysteine-rich for example. Although overcoming some of the caveats observed in 2-DE approach, shotgun proteomics using LC

57 methods also suffer from a limited dynamic range, loss of protein isoforms and PTMs information as well as bioinformatic challenges to infer protein/peptide identification from the complex amount of MS data generated.

3.2.4. Mass spectrometry & Bioinformatics

Mass spectrometry (MS) is the core component of proteomics, allowing the sensitive and rapid identification and quantification of thousands of polypeptides through mass over charge (m/z) measurements. This chapter focuses on bottom-up strategies, involving the liquid or in-gel digestion of protein samples prior to MS. Trypsin is the enzyme of predilection as it cleaves very specifically at C-terminal ends of and arginine residues except when they precede a proline, resulting in multiply charged peptides easily ionizable. Following digestion, peptides usually undergo C18 desalting or fractionation by high-pressure (HP)-LC. All potentially interfering compounds must be removed prior MS, including stains from gel-based procedures

(i.e. silver-staining), detergents or salts.

Driven by the inextricable complexity of proteomes, technical limits of MS instrumentation are constantly pushed, with the development of multiple ion sources, analyzers or detectors, the three main components of mass spectrometers. In the ion source, analytes are ionized and transferred from their condensed to gas phase. Two types of soft ionization sources are generally used in proteomics, matrix-assisted laser desorption/ionization (MALDI) [239] and electrospray ionization (ESI)[240], both able to convert polar molecules such as proteins or peptides into gas phase ions. Then, the mass analyzer separates peptides according to their m/z and finally the “detector” records the abundance of each ion to create a signal converted into a mass spectrum (m/z versus signal). Different mass analyzers types are available in proteomics – the time of flight (TOF), ion trap (IT), quadrupole (Q), Fourier transform ion cyclotron resonance

(FTICR) or Orbitrap, which differ in their sensitivity, mass accuracy and resolution performances.

Whereas single-stage mass spectrometers (i.e. MALDI-TOF) allow the simple measurement of

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polypeptides’ molecular mass, hybrid mass spectrometers (i.e. TOF-TOF, LTQ-OT) enable the determination of protein amino acid sequence, expression level and structural features (i.e. PTM sites) using multiple stage MS fragmentation (MSn). Protein identification and quantification are generally obtained by tandem mass spectrometry (MS/MS). In this mode, a first analyzer acts as a filter to select specific ions which are transmitted to a second analyzer that serves as a collision cell where ion fragmentation is induced by an inert gas. Collision induced dissociation (CID) using argon is the most common fragmentation process. For instance, the linear trap quadrupole

(LTQ)-Orbitrap is one of the most performant and recent instrument commercialized, combining the MSn capability of the linear ion trap (LTQ) with the high resolution and mass accuracy of the

FT Orbitrap [241-243] (Figure 19).

Figure 19. Schematic representation of a tandem mass spectrometer: the LTQ-Orbitrap XL. Precursor ions from an ESI source are first analyzed in MSn by a linear ion trap (LIT) mass analyzer, transferred to a C-trap for ion storage and further injection in the High energy C-trap dissociation (HCD) cell as well as in the high resolution Orbitrap mass analyzer. CID mode performed in the LIT is generally used for peptide identification whereas HCD fragmentation mode, which generates smaller fragments, is typically used for the analysis of isobaric tag (TMT, iTRAQ) reporter ions, Image from Thermo Scientific.

Several search algorithms, including Mascot [244] or Phenyx, have been developed to enable the assignment of peptide sequences by comparing experimental MS/MS spectra and theoretical MS fragmentation patterns of tryptic peptides present in the database, referred to as peptide fragment fingerprinting (PFF). Typically, theoretical masses are obtained after in silico

59 protein digestion and peptide fragmentation of all proteins present in a database. MS has been driven by the complete sequencing of the followed in 2008 by the complete annotation of its protein counterpart in the manually curated database UniProtKB/Swiss-Prot.

However peptide or protein sequences can be absent from databases, mainly due to the huge range of post-transcriptional and post-translational modifications. In that case, de novo sequencing can be performed manually by determining mass intervals between two peaks in

MS/MS spectra, corresponding each to one amino acid. As peptide/protein identification is a probability based process, false discovery rates (FDR) can be calculated to estimate the rates of mistakenly identified proteins and should generally be kept below 1% at the peptide or protein level [245]. Finally, bioinformatics tools can also be useful to extract biological information from protein lists, through gene ontology classification or KEGG pathways analyses for instance, using webtools such as DAVID [246, 247].

3.2.5. Quantitative proteomics

The sole identification of extensive datasets of proteins associated to CSF (i.e. 2594 proteins) [248], specific brain region including cortex (i.e. 812 proteins) [249] and to subcellular compartments such as cortical LBs (i.e. 296 proteins and 40 proteins) [230, 231] or nigral neuromelanin granules (i.e. 72 proteins) [232], has been essential for a better characterization of the CNS and its specific functions. However, to gain more insights into disease pathogenesis, quantitative proteomic data are needed to determine the specific set of proteins exhibiting differential expression levels in healthy versus pathological states. Relative quantification has traditionally been performed by 2-DE or DIGE, followed by staining and image analysis using softwares such as ImageMaster 2D platinium (GE Healthcare) to identify differences in gel patterns (Figure 20 A). CSF and serum were profiled by 2-DE, allowing the detection of a few differential proteins (i.e. complement c3) between control and PD patients that could serve as

PD biomarkers [250-252]. Several 2-DE studies of human brain tissues specifically analyzing the

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SN were conducted, allowing a deeper understanding of PD pathogenesis through the identification of several novel abnormalities in the proteome of PD patients including CNDP2 overexpression [226-228]. 2-DE was also proved useful for elucidating some of the PTMs associated with PD. For example, three 2-DE studies by Choi et al. demonstrated oxidations in multiple proteins previously linked to PD including the chaperone DJ-1, superoxide dismutase

Cu/Zn, as well as UCH-L1 in the frontal cortex of PD patients compared to controls [253-255].

Although providing access to a range of PTMs and protein isoforms, this procedure might not be the best-suited for the rapid analysis of complex samples, suffering principally from a lack of automatization and a limited dynamic range together with reproducibility, resolution and sensitivity issues.

Alternatively, high throughput shotgun quantitative proteomics using multidimensional

LC can be used to analyze complex mixtures. This approach mainly relies on stable isotope labeling of proteins or peptides, which consists in the introduction of a mass tag to differentiate identical peptides from several samples. Differentially labeled peptides (i.e. heavy or light) can be discriminated in MS owing to a mass shift and relatively quantified by plotting the intensity ratio of the different forms (i.e. heavy over light) of the same peptide sequence. Peptide labeling has been achieved through various methods including metabolic labeling for cultured cells

(SILAC), O18 labeling, or isotope-coded affinity tags (ICAT) which tags cysteine-containing residues. Peptide identification and relative quantification are here performed sequentially through tandem MS/MS and MS, respectively. Using ICAT, 119 proteins were found to exhibit changes in their relative expression in mitochondrial fractions obtained from the SNpc of PD compared to controls, and the observed decrease in mortalin was further confirmed in a cellular

PD model [229]. Isobaric tagging technologies either using tandem mass tags (TMT) [224, 256] or isobaric tags for relative and absolute quantification (iTRAQ) [257, 258] have allowed in a single

MS/MS experiment, simultaneous peptide identification and quantification in multiple samples

(up to six for TMT6), (Figure 20 B). Isobaric tags contain a reactive group attaching to primary

61 amines (N-term or Lysine), a reporter group that serves for quantification in MS/MS, a cleavable linker enabling the release of the reporter upon CID and a mass normalization group balancing mass shifts to keep the total mass of the tags constant (isobaric). Thus, labeled identical peptide sequences co-elute and appear at the same mass in MS scan, but give rise to low mass reporter signature ions upon CID fragmentation in MS/MS mode (i.e. between 126 and 131 m/z for TMT-

6). This robust method has been one of the most beneficial for the analysis of body fluids and tissues in PD. Shotgun proteomic quantitative analyses of CSF have allowed the detection of various changes in the CSF composition of PD patients, reviewed in [259]. Interestingly, Abdi et al. found 72 proteins - including ceruloplasmin or apolipoprotein H, uniquely associated to PD compared to AD, dementia with LBs and control patients differentially labeled with iTRAQ-4

[257]. No single biomarker has already been validated for PD but a combination of them may prove useful as demonstrated with a panel of eight proteins (i.e. tau, amyloid β-42, β-2 microglobulin, interleukin-8, vitamin D binding protein, apolipoproteins A-II and E and BDNF) able to distinguish PD from AD and control patients with high sensitivity and specificity [260]. In this thesis project, we analyzed SN tissues by TMT sixplex (TMT-6) to label PD (n=3) and control patients (n=3), allowing the identification and quantification of about 1795 proteins. Some of them were differentially expressed between the two groups, pointing towards previously known and novel pathological PD mechanisms (see Chapter V). The comprehensive analysis of specific

PTMs known to be important for PD, such as oxidation, nitration, phosphorylation, glycosylation or ubiquitination can also be addressed, as reviewed in Chapter II. Generally, proteomes of interest are specifically enriched (see “sample separation section”) before being analyzed by MS quantitative techniques using stable isotope tags. Alternatively, peptides with defined PTMS can be targeted based on their MS fragmentation characteristics (i.e. neutral loss, multiple reaction monitoring MS modes). Finally, label-free quantification methods based on spectral counts seems to be promising as well [261, 262]. Importantly, absolute quantification can be obtained

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through AQUA method, with the spiking of a known quantity of an isotope-labeled peptide as an internal standard, followed by single reaction monitoring (SRM) MS analysis [263].

A. Two-dimensional gel analysis (2DE) IEF Control pI 3 10 PD MALDI TOF-TOF 4800

PD Control Spot

PAGE MW - Spot Spot quantifications SNpc tissues excisions identifications SDS 2D-Image Master PD patients (n=3) Controls (n=3) Protein MS/MS extraction & analysis solubilisation B. Shotgun proteomic using TMT6 Digestion Ctrl 1 Ctrl 2 Ctrl 3 2D peptide separation ↓ 126 ↓ 128 ↓ 130 Protein 1) OFFGEL fractionation Samples identifications PD 1 PD 2 PD 3 pooling & ↓127 ↓ 129 ↓ 131 2) RP – LC C18 quantifications

LTQ-orbitrap

Figure 20. Quantitative proteomic workflows. After tissue dissection, protein extracts are prepared and can either be submitted to a 2-DE workflow (A) or digested and submitted to a shotgun proteomic approach (B). In the 2-DE approach protein spots are compared between gels and quantified using image analysis softwares, and spots of interest are analyzed by MS for identification. In the shotgun approach, differentially labeled peptide samples are identified and quantified by MS/MS.

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4. PROJECT PRESENTATION

Current unmet needs in PD research include the elucidation of PD pathogenic mechanisms and the identification or reliable biomarkers for diagnosis, both prerequisites for the establishment of neuroprotective therapies. The substantia nigra (SN) is thought to be the lesional core of PD pathology, exhibiting a massive neurodegeneration of dopaminergic neurons along with the occurrence of Lewy bodies (LB). The resulting DA depletion underlies the main motor disabilities. Understanding the specific vulnerability of the SN as well as the pathological molecular pathways and biological events triggering neuronal death in the SN of PD patients are primary goals in PD.

We postulated that the elucidation of the complex proteome alterations occurring in the

SN of PD patients compared to controls may allow the exploration of previously known or novel pathogenic mechanisms engaged in PD pathogenesis. In addition, it may ultimately provide a new source of PD-specific therapeutic targets and biomarkers for the treatment and prevention of PD.

We hypothesized that:

 PD is a widespread brain “proteinopathy” not restricted to α-SYN, in which several proteins

experience altered processing at different levels (i.e. expression, PTMs) either as a cause or

a consequence of the neurodegenerative process.

 The investigation of human autopsy samples from PD patients is an inescapable source of

information and an avenue of choice to detect PD specific abnormalities.

 Proteomic strategies may be particularly well-suited to capture the complex nature of PD,

allowing the comprehensive analysis of the pathological effector molecules themselves.

 After neuronal death, some of these alterations may be released in the surrounding

biofluids (i.e. CSF, blood, urine etc). If detectable, these neuropathologically-derived

proteins could serve as biomarkers to help in the early, specific and sensitive PD diagnosis.

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The primary goals of the project are to 1) characterize the SN proteome to improve our understanding of SN function and specificities and 2) identify a subset of differentially expressed proteins, potentially pathogenic, in the SN of PD patients. A secondary goal, as an extension of this study is to test some of the validated molecules of interest as neuropathologically-derived biomarkers of PD in the CSF of PD patients.

This thesis manuscript is divided into 5 parts:

 Chapter II consists in a review of the current literature on proteomics applied to PD

research, published in Journal of proteomics (2009). The paper covers some advances in PD

pathogenesis and biomarker research driven by human proteomics. Of note, it provides an

evaluation of the various proteomic strategies available to examine protein expression and

modifications in different samples.

 Chapter III presents an editorial published in Expert reviews of proteomics (2011) proposing

to reappraise the validity of human samples such as biological fluids from living patients or

brain derived autopsy tissues in PD proteomic research.

 Chapter IV is an original article published in Journal of proteomics (2012) presenting a 2-DE

proteomic comparison of the SN in patients with PD and age-matched controls. The role of

the differential protein CNDP2 is further discussed, together with its potential as a

biomarker.

 Chapter V presents a high-throughput quantitative proteomic profiling of the SN in PD

patients compared to non-neurological controls, using an isobaric chemical labeling

technique termed tandem mass tag (TMT). This study provides the most extensive

assessment of nigral proteome, and a catalogue of proteome alterations. The chapter will

be submitted for publication.

 Chapter VI provides a general discussion of the results obtained in this thesis and proposes

a few perspectives to the project.

65

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Chapter II

Proteomics in human Parkinson’s disease

Parkinson’s disease (PD) is an exceedingly complex and enigmatic condition which has been the matter of intensive investigations during the last decades. In particular, the precise etiopathological mechanisms underlying the selective degeneration of vulnerable dopaminergic neurons in the substantia nigra (SN) of PD patients are still unknown. After recapitulating the current knowledge on PD pathology, the review presented in this chapter provides an update on proteomics contribution to the field of PD, through the profiling of human samples (CSF, brain tissues). Importantly, the review critically addresses proteomics limitations but also depicts the unique potential offered by proteomics to unravel PD pathogenesis.

I entirely wrote this review published in 2009 in Journal of proteomics (IF=5.1).

JOURNAL OF PROTEOMICS 73 (2009) 10– 29

available at www.sciencedirect.com

www.elsevier.com/locate/jprot

Review

Proteomics in human Parkinson's disease research

Virginie Lickera, Enikö Kövarib, Denis F. Hochstrasserc, Pierre R. Burkharda,d,⁎ aNeuroproteomics Group, University Medical Center, Faculty of Medicine, University of Geneva, Switzerland bDepartment of Psychiatry, Geneva University Hospitals, Switzerland cDepartment of Genetics and Laboratory Medicine, Geneva University Hospitals, Switzerland dDepartment of Neurology, Geneva University Hospitals, Geneva, Switzerland

ARTICLE INFO ABSTRACT

Article history: During the last decades, considerable advances in the understanding of specific mechanisms Received 8 April 2009 underlying neurodegeneration in Parkinson's disease have been achieved, yet neither Accepted 8 July 2009 definite etiology nor unifying sequence of molecular events has been formally established. Current unmet needs in Parkinson's disease research include exploring new hypotheses Keywords: regarding disease susceptibility, occurrence and progression, identifying reliable diagnostic, Parkinson's disease prognostic and therapeutic biomarkers, and translating basic research into appropriate Lewy body disease-modifying strategies. The most popular view proposes that Parkinson's disease Substantia nigra results from the complex interplay between genetic and environmental factors and Proteomics mechanisms believed to be at work include oxidative stress, mitochondrial dysfunction, Protein excitotoxicity, iron deposition and inflammation. More recently, a plethora of data has α-synuclein accumulated pinpointing an abnormal processing of the neuronal protein α-synuclein as a pivotal mechanism leading to aggregation, inclusions formation and degeneration. This protein-oriented scenario logically opens the door to the application of proteomic strategies to this field of research. We here review the current literature on proteomics applied to Parkinson's disease research, with particular emphasis on pathogenesis of sporadic Parkinson's disease in humans. We propose the view that Parkinson's disease may be an acquired or genetically-determined brain proteinopathy involving an abnormal processing of several, rather than individual neuronal proteins, and discuss some pre-analytical and analytical developments in proteomics that may help in verifying this concept. © 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction ...... 11 2. Neuropathology of Parkinson's disease: the enigma of progression ...... 11

Abbreviations: ALS, amyotrophic lateral sclerosis; α-SYN, α-synuclein; AD, Alzheimer's disease; CNS, central nervous system; CSF, cerebrospinal fluid; DA, dopamine; DUB, deubiquinating enzyme; 2-DE, two-dimensional electrophoresis; HE, hematoxylin–eosin; LB, Lewy body; LC, locus ceruleus; LCM, laser capture microdissection; LD, levodopa; LN, Lewy neuritis; LP, lumbar puncture; MPTP, 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine; NM, neuromelanin; PD, Parkinson's disease; PTMs, post-translational modifications; ROS, reactive oxygen species; SNpc, substantia nigra pars compacta; TMT, tandem mass tags; UB, ubiquitin; UPS, ubiquitin–proteasome system; VTA, ventral tegmental area. ⁎ Corresponding author. Department of Neurology, Geneva University Hospitals, 4, rue Gabrielle-Perret-Gentil, 1211 Geneva 14, Switzerland. Tel.: +41 22 372 83 09; fax: +41 22 372 83 32. E-mail address: [email protected] (P.R. Burkhard).

1874-3919/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2009.07.007 JOURNAL OF PROTEOMICS 73 (2009) 10– 29 11

3. Selective neurodegeneration ...... 12 4. Lewy body ...... 13 4.1. Structure and composition ...... 13 4.2. Alpha-synuclein ...... 13 5. Potential mechanisms underlying degeneration ...... 14 5.1. Oxidative stress and mitochondrial dysfunction ...... 14 5.2. Protein aggregation and the ubiquitin–proteasome system ...... 15 5.3. Many unsolved issues ...... 15 6. Proteomics applied to Parkinson's disease research ...... 17 6.1. Proteomics in animal models of PD ...... 17 6.2. Proteomics in human PD ...... 17 6.2.1. CSF protein profiling ...... 17 6.2.2. Brain tissue protein profiling ...... 18 6.2.3. Cellular and subcellular proteomes ...... 19 7. Potential avenues and conclusion ...... 20 7.1. Quantitating protein expression ...... 21 7.2. Examining protein modifications ...... 22 7.2.1. Oxidation, nitration and S-nitrosylation ...... 22 7.2.2. Phosphorylation ...... 22 7.2.3. Ubiquitination ...... 24 7.3. Studying protein miscleavage ...... 24 Acknowledgments ...... 24 References ...... 24

1. Introduction and postural instability. Since the pioneering work of Fritz Heinrich Lewy in 1912 and Constantin Trétiakoff in 1919, the Parkinson's disease (PD) is the most common neurodegenera- neuropathological lesions at the basis of typical, sporadic PD tive movement disorder, affecting 1–2% of the population over have been extensively studied and comprehensively defined the age of 60 years [1] and over 6 million people worldwide. [7,8]. Classically, there is a progressive loss of neurons in Recent epidemiological projections herald a continuously selected pigmented and non-pigmented brainstem and cere- increasing prevalence along with populations' aging and it is bral nuclei, particularly in the substantia nigra pars compacta anticipated that the number of affected individuals may (SNpc) in the midbrain and the locus ceruleus (LC) in the pons. double in the most populous nations within the next two There is also a mild degree of astrocytic and microglial decades [2]. As a result, PD represents a growing socio- reaction. Remaining neurons show inclusions, known as economic burden on societies and a major public health pale bodies and Lewy bodies (LBs, Fig. 1a) when located in challenge for the future, the more so as, despite extensive their cytoplasm, and Lewy neuritis (LNs, Fig. 1d) in their research, neither the cause nor the mechanisms underlying neuronal processes, which have been shown using immuno- α the condition have been firmly established thus far. Con- chemistry techniques to be notably composed of -synuclein α sequences resulting from this uncertainty include poor ( -SYN) (Fig. 1b, f, h) [9]. The temporal and topographical diagnostic accuracy, absence of valid biomarkers and difficul- progression of PD pathology in the central (CNS) and, to a ties to elaborate therapeutic interventions that might sig- lesser extent, the peripheral autonomic nervous system, has – nificantly influence disease progression. In this review article, been recently refined [10 13]. According to these studies, PD α we make the assumption that modern proteomic technologies pathology, defined as -SYN positive aggregates, appears to may offer new insights into the pathogenesis of PD and, while follow a non-random, caudo-rostral progression within the broad reviews have already been published in this field [3–5], CNS, starting in the dorsal motor nucleus of the vagus nerve in we will focus on proteomics as a tool to approach pathogen- the brainstem, in the olfactory bulb and anterior olfactory esis of sporadic PD in humans. nucleus. Lesions then become more and more pronounced in affected areas while progressing rostrally and involving subsequent structures in a stereotyped and selective manner 2. Neuropathology of Parkinson's disease: the through what Braak and colleagues have defined as six enigma of progression neuropathological stages of PD, the first two stages corre- sponding to presymptomatic PD and the next four to sympto- Since being formally recognized as a distinct morbid entity in matic PD. During this pathological process, selective 1817 by James Parkinson in his seminal “Essay on a Shaking structures of the brainstem, temporal mesocortex and even- Palsy” [6], PD has been essentially regarded as a disorder of tually neocortex are progressively involved. Consequently, motor function, producing a variable, asymmetric and pro- involvement of the SNpc, which occurs during PD's Braak gressive combination of resting tremor, bradykinesia, rigidity stage 3, is just another step in a much larger, multisytem 12 JOURNAL OF PROTEOMICS 73 (2009) 10– 29

specific brain regions to exhibit Lewy pathology, irrespective of the timing of events. According to this view, there is no caudo-rostral progression and the common involvement of the SNpc, the LC and the dorsal motor nucleus of the vagus nerve, either alone or in various combinations, reflects the individual likelihood and vulnerability of these structures to develop Lewy deposits. From a research point of view, whatever the mode of progression, these neuropathological considerations imply that information extracted from human samples, taken at a certain time point during PD progression, will, somewhat artifactually, offer an instantaneous view of a constantly evolving pathology and may not necessarily reflect the complex dynamics of the whole degenerative process.

3. Selective neurodegeneration

Whereas the etiology of sporadic PD remains unknown, many pathogenic mechanisms have been shown to be likely at work in the SNpc of PD patients, including oxidative stress and production of reactive oxygen species (ROS) in excess, mitochondrial dysfunction and energy production imbalance, excitotoxicity mediated by increased glutamatergic stimula- tion, excessive deposition of reactive iron and abnormal iron– neuromelanin (NM) interaction, inflammation and immune- mediated reactions, defects in neurotrophic factors, and apoptosis, among others [17]. More recently, an abnormal cellular protein processing and dysfunction of the ubiquitin– proteasome system (UPS) have been suggested to be central to LB formation and neurodegeneration. It is likely that more than one of these mechanisms is involved, yet the exact combination and succession of which remain unclear. The nigrostriatal pathway is formed by DA-containing neurons projecting to the striatum (i.e., putamen and caudate nucleus) whose cell bodies are confined in a densely populated – “ ” Fig. 1 Histological lesions of Parkinson's disease: classical SN subdivision individualized as the pars compacta. The SNpc “ ” or brain stem-type Lewy bodies in the substantia nigra acts as a modulator of motor function via a dual effect upon the – pars compacta (a c); Lewy neurites (d); pale bodies (e and f) basal ganglia loops, through dopamine receptor D1-mediated and cortical Lewy bodies in the entorhinal cortex (g and h). activation along the direct pathway and D2-mediated inactiva- (a, e and g: HE staining; b, d, f and h: immunohistochemistry tion of the indirect pathway, to promote voluntary and inhibit α with antibody anti- -synuclein; c: immunohistochemistry involuntary movements. In PD, the extensive neuronal degen- – – with antibody anti-ubiquitin. Scale bar: a c and e h: 20 µm, d: eration of this dopaminergic pathway alters the balance 60 µm). between both loops resulting in the typical features of PD, in particular bradykinesia. While PD neurodegeneration is not restricted to dopaminergic systems, the reasons why a selected pathology, which involves not only motor but also non-motor, population of DA neurons is primarily targeted while others autonomic, limbic and cognitive systems. It is important to remain marginally or not at all affected, is an important issue to appreciate that, in this scenario, neurodegeneration is be addressed. Indeed, neurodegeneration of the DA neurons initiated a considerable time before symptoms become does not seem to occur randomly or diffusely within the SNpc or detectable — probably 5 to 10 years — and the disease the other DA systems. In fact, nigral lesions are regionally highly clinically starts when about 50 to 70% of nigral neurons have selective with the ventrolateral and caudal tiers being primarily already been lost [14]. Thus, an early clinical diagnosis of PD is and more severely affected, whereas the dorsomedial tier is necessarily made at an advanced pathological stage. more affected during normal aging [18]. Although age constitu- However, the concept proposed above has been recently tes the most important risk factor for the development of PD, the challenged mostly because of the lack of correlation between processes inducing age-related dopaminergic neuronal loss the Braak staging system and the clinical severity of PD, seem to be anatomically distinct from those underlying PD. casting doubt about its predictive validity [15,16].Asan This heterogeneity and selective vulnerability of DA — and also alternative, it has been hypothesized that the regional pattern non-DA — neurons has been hypothesized to result from of α-SYN deposition may reflect the relative propensity of morphological specificities. Indeed, a relatively low number of JOURNAL OF PROTEOMICS 73 (2009) 10– 29 13

DA neurons (about 450,000 in adult human SNpc) provide Altogether, neurodegeneration of PD does not occur at massive striatal innervations through disproportionately long, random and appears to be topographically patterned not only thin and branched axonal projections, compared to a relatively throughout the CNS but also within affected structures. Under- small cell body, which are poorly or not myelinated. This type of standing the basis of this selectivity, which is likely to somehow neuron may need higher energy production to achieve basic reflect pathogenesis [23], may be of crucial importance. functions, particularly axonal transmission, and may be more vulnerable to any form of insult [19]. Compared to most brain structures, DA neurons are more exposed than others to 4. Lewy body oxidative stress. Their elevated rate of oxygen and calcium metabolism, low levels of antioxidants and high iron content are Lewy inclusions, the pathological hallmark of PD, are found in the all factors inducing higher rate of damaging ROS. Thus, their cytoplasm of surviving neurons of the SNpc but also in non- specific physiology may also hold the key of their vulnerability. dopaminergic neurons such as the LC or the cortex. They exhibit a For example, DA neurons maintain an autonomous activity variable morphology, developing as isolate or multiple, spherical relying on L-type calcium channels, whereas other neurons use or elongated, 8 to 30 µm in diameter, LBs in cell bodies, or spindle- sodium channels [20]. This reliance implies an increased and like LNs in neuronal processes. Composition of LBs, molecular sustained energy consumption to sequester and pump calcium mechanisms of aggregation, biological role and implication in the out of the cell. Ultimately, this imposes an increased metabolic pathogenic processes underlying PD are still unclear, in spite of stress on mitochondria, accelerating cellular ageing and death the identification of α-SYN as an important component [9]. [20]. In addition, DA itself may predispose, induce or facilitate Importantly, α-SYN inclusions are not specific for PD and occur degeneration. Its chemical instability and auto-oxidation cap- also in other neurological conditions, tentatively classified as ability, occurring both spontaneously and enzymatically, may “alpha-synucleinopathies”, which include besides PD, dementia potentially lead to ROS production. Two populations of DA with LBs, the LB variant of Alzheimer's disease (AD), multiple neurons can be observed in the mesencephalon, those contain- system atrophy, and neurodegeneration with brain iron accu- ing NM, a major DA oxidation by-product [21], and those mulation type I, among others. without. A correlation between the vulnerability of nigral neurons and NM pigmentation has been established. Indeed, 4.1. Structure and composition NM-containing neurons which comprise not only DA neurons of the SNpc but also noradrenergic neurons of the LC seem to be In PD, two variants of intracytoplasmic LBs can be distin- more prone to degenerate in PD [22].Thus,notonlyDAbutalso guished: the classical brainstem type found within the NM itself could be cytotoxic and contribute to oxidative stress perikaryon of DA nigral neurons and a less common type through its interaction with iron and other reactive metabolites found in cortical neurons. At light microscopy using hematox- [22]. ylin/eosin (HE) staining, classical LBs typically exhibit a dense Among DA neurons, another pattern of regional vulnerability core surrounded by a clearer halo (Fig. 1a). Cortical LBs are less has been observed, with some populations showing less or no eosinophilic and present with a more homogeneous aspect loss at all [23–25]. For example, adjacent midbrain DA popula- lacking the distinctive core and halo (Fig. 1g). The greater tions within the ventral tegmental area (VTA) and their sensitivity of immunostaining analysis has allowed an easier projections to the ventral striatum exhibit a relative resistance identification of LBs [31] and demonstrated that α-SYN labels to PD-related degeneration. In contrast to SNpc, VTA neurons specifically the halo whereas ubiquitin (UB) staining yields a contain little NM, indicating less DA-related oxidative stress more diffuse labelling (Fig. 1b, c). Other structures weakly during neuron life span [26]. Furthermore, it is also possible that stained with HE but clearly positive for α-SYN and UB are also neuron surrounding influences their fate critically. Indeed, detected, including pale bodies (Fig. 1e, f). Some data suggest susceptible neurons tend to lie in calbindin-poor regions of the that pale bodies correspond to LBs at an early stage of SNpc [23], known as nigrosomes, whereas neurons in calbindin- development [32,33]. Ultrastructurally, classical LBs seem to enriched regions, known as the nigral matrix and containing be composed of fibrils radiating out from a central core of neuron terminals issued from the striatum, are spared. The dense granular material whereas the cortical type is composed significance of this topographical distinction is unknown. almost exclusively of circular or oval material [34].Our Finally, and at variance with the observations supporting the knowledge regarding the biochemical composition of LBs has neuron body as the main target of degeneration in PD, it has also evolved with more sophisticated immunohistochemical tech- been proposed that the loss of nigral neurons may result from a niques, showing that LBs were notably positive for many “dying back” process whereby striatal nerve terminals are the compounds besides UB and α-SYN. In fact, the use of various primary target of the degenerative process [27]. Indeed, the non-proteomic techniques has led to a list of nearly 80 proteins degree of terminal loss in the striatum appears to be dispro- identified thus far within LBs (Table 1). portionally more pronounced compared to the severity of SNpc neuron loss [28,29]. Whereas this scenario may explain the 4.2. Alpha-synuclein topology of neuronal loss in the SNpc which is somatotopically connected to the putamen, it seems at odds with the notion of α-SYN belongs to the synuclein family of proteins composed of α, structural compensatory mechanisms that are believed to occur β and γ-synuclein, which are extensively expressed in the brain. in the striatum to attenuate the effects of reduced DA Although the exact physiological functions of α-SYN remain transmission, one of which relying on synaptogenesis, i.e. uncertain, it may be involved in neurotransmission by regulating sprouting of new terminals by surviving neurons [30]. synaptic vesicles size, recycling and plasticity [35]. α-SYN is a 14 JOURNAL OF PROTEOMICS 73 (2009) 10– 29

Table 1 – List of proteins identified in Lewy bodies using non-proteomic, essentially immunohistochemistry staining techniques.

Advanced glycation Complement proteins Immunoglobulins (IgGs) P35nck5a Synaptic vesicle-specific endproducts (C3d, C4d, C7, C9) protein αβ-crystallin Chondroitin sulfate Phosphorylated IkBa P38 Synaptophysine proteoglycans α-synuclein Chromogranin A Heme oxygenase-1 p62/sequestosome 1 Synaptotagmine α2-macroglobulin Cullin-1 Histone deacetylase 4 Parkin Tau Agrin Cyclin B Leucine-rich repeat kinase 2 Phospholipase C-δ Tissue transglutaminase (LRRK-2) Amyloid precursor Cyclin-dependent kinase 5 Lipids PTEN-induced Torsin A protein (APP) kinase 1 (PINK1) Apolipoprotein J/clusterin Cytochrome c Microtubule-associated Prolyl- Pin Tropomyosine proteins (MAP-1, 1b, 2, 5) 1 26S proteasome ATPase DJ-1 Multicatalytic proteinase Retinoblastoma protein Basic fibroblast growth Dorfin mxA protein ROC1 Tubulin polymerization factor promoting protein/p25 β-TrCP Extracellular signal- NEDD8 sept4/H5 Tyrosin hydroxylase regulated kinases C terminus of 14-3-3 protein Neurofilaments SIAH-1 Ubiquitin Hsp70-interacting protein Calcium/-dependent G-protein-coupled receptor NFκB, IκBa Sphingomyelin Ubiquitin activating protein kinase II kinase 5 enzyme (E1)

Calbindin D28K Gelsoline-related amyloid NUB1 Cu/Zn superoxide Ubiquitin conjugating protein Finnish type dismutase enzyme UbcH7 (E2) Centrosome/aggresome-related Glyceraldehyde-3-phosphate omi/HtrA2 Mn superoxide Ubiquitin C-terminal proteins dehydrogenase dismutase hydrolase (UCH-L1) CHIP Heat shock proteins (HSP 27, pael-R Synphilin-1 Vesicular monoamine 40, 60, 70, 90, 110) transporter 2

According to Shults CK, 2006, and Wakabayashi K et al., 2007. Genetic defects in proteins highlighted in bold have been reported to cause or increase the susceptibility for Parkinson's disease.

highly conserved 140-aminoacid presynaptic protein of ~19 kDa mechanism that might be common to sporadic PD as well, in view originally identified in humans as the non-amyloid beta compo- of the recent finding that some polymorphisms of the promoter nent (NAC) of AD amyloid plaques [36].Itsstructurecanbe region of the SNCA gene may increase the risk of PD in the general divided into three regions. First, a 60-aminoacid N-terminal population [44–46]. In addition, many factors that can promote α- region containing 11 imperfect repeats with highly conserved SYN aggregation have been identified. For example, α-SYN was KTKEGV motives, is predicted to form amphipatic alpha-helix shown to carry post-translational modifications (PTMs) such as typical of apolipoprotein-like-class-A2 which mediates binding to phosphorylation (at Ser129) and ubiquitination (at Lys12, Lys21 phospholipid vesicles [37]. Second, a central region containing the and Lys23) that could contribute to its toxicity [47]. Deleterious NACsequencefromresidue61to95whichappearstobeessential interactions between α-SYN and environmental agents or other for aggregation [36]. Third, a C-terminal portion, less conserved, proteins could also dramatically accelerate the rate of α-SYN containing many acidic residues which confer to the molecule a fibrillogenesis in vitro and impairing the process of degradation of low pI. Natively, α-SYN presents in a soluble, unfolded state in the abnormal protein and/or increase oxidative stress [48,49]. the cytoplasm but can be found in more ordered states such as α- helical conformation in the presence of lipid membranes or as β- sheets in fibrils composing LBs [38,39]. 5. Potential mechanisms underlying Involvement of α-SYN in PD pathogenesis derives from the degeneration identification of missense mutations (A53T and A30P) in the SNCA gene in early-onset familial PD kindreds with autosomal 5.1. Oxidative stress and mitochondrial dysfunction dominant inheritance [40,41]. Interestingly, predictions suggest that these N-terminally located mutations could disrupt the α- Post-mortem studies have consistently implicated oxidative helix and extend the β-sheet structure, which is thought to be damage to lipids, proteins, and DNA in the pathogenesis of PD, involved in the self-aggregation of proteins and the formation of as observed in the SNpc of sporadic PD brains [50]. The source amyloid-like structures. Perhaps more importantly, duplications of this increased oxidative stress is uncertain but may include and triplications of the α-SYN gene SNCA were also reported in mitochondrial dysfunction, increased dopamine metabolism, autosomal dominant forms of PD, resulting in increased levels of reactive iron deposition and impaired antioxidant pathways. wild-type α-SYN in the absence of other known mutations [42,43]. In fact, much evidence suggests a major role for defects in These data suggest that abnormal intracellular accumulation of complex I of the respiratory chain leading to decreased ATP wild-type α-SYN is sufficient to induce neurodegeneration, a production as well as damage caused by excess production of JOURNAL OF PROTEOMICS 73 (2009) 10– 29 15

ROS. Consistent with this hypothesis, altered complex I somes. Under non-physiological conditions, such as molecu- activity was observed in the SNpc of sporadic PD patients lar crowding, oxidative stress, PTMs or modifications of the [51]. In addition, several observational and epidemiological protein primary structure, soluble monomers and oligomers of studies suggest that pesticides and other environmental toxins native or non-native polypeptides may self-assemble, poly- that inhibit complex I are involved in the pathogenesis of merize and coalesce into giant intracellular inclusions, such as sporadic PD [52]. For example, 1-methyl 4-phenyl 1,2,3,6- LBs in PD [59]. In fact, it has been suggested, based on many tetrahydropyridine (MPTP) was initially identified as an similarities between both structures, that aggresomes and LBs accidental by-product during the illicit manufacture of a may be created following the same cytoprotective process of synthetic opiate in Northern California in the early 80s, and protein sequestration. Whereas aggresomes may be cleared by drug users who injected MPTP developed an acute syndrome enhanced proteasome activity and autophagy, LBs may not, closely resembling PD [53]. The selectivity of MPTP for for reasons that are yet unclear [60]. Eventually, LBs and LNs dopaminergic neurons relates to its conversion in astrocytes may impair critical intracellular processes, notably axonal by monoamine oxidase B to the active metabolite, 1-methyl-4- transport, synaptic plasticity and neurotransmission, and phenyl pyridinium (MPP+), which is taken up in DA neurons eventually promote degeneration. through the dopamine transporter, where it inhibits complex I Because the UPS is a major pathway mediating the and ultimately leads to cell death [27]. Similarly, the widely degradation of abnormal cellular proteins [61], it has been used herbicide paraquat, the insecticide rotenone and the proposed that its dysfunction may be a key factor in the fungicide maneb (manganese ethylenepistithiocarbamate) all pathogenesis of PD and LB formation (reviewed in [62]) either inhibit complex I and induce a selective loss of nigrostriatal primarily or secondary to an impairment of the capacity of the neurons in rodents [54]. In rotenone-treated rats, LB-like UPS to handle an overwhelming quantity of altered proteins. intraneuronal filamentous inclusions containing α-SYN and In particular, failure of the 26S proteasome may be critical [63]. UB have been observed [55]. In addition, a connection between For example, UB has been observed to concentrate in LBs α-SYN and complex I is supported by the observation that α- where it colocalizes with chaperones and UPS components SYN knockout mice are resistant to MPTP, whereas α-SYN that normally take part in the protein quality control transgenic mice show enhanced toxicity [56], suggesting that machinery and clearance of abnormal proteins in the cell. To α-SYN is required for mediating the downstream effects of further support this view, it has been recently shown that complex I inhibition. These findings are consistent with systemic administration of the proteasome inhibitors epox- alterations in complex I being central to the pathogenesis of omicin or PSI in rats induces a gradually progressive, levodopa sporadic PD and support environmental factors as main (LD)-responsive, form of parkinsonism similar to PD in contributors to PD pathogenesis. However, the role of mito- humans [64]. The central role of the UPS in PD pathogenesis chondrial defects and oxidative stress has also been chal- is, however, still a matter of disagreement [65]. lenged by several authors arguing that these alterations may merely reflect the unspecific and additional effects of aging [57] 5.3. Many unsolved issues or the consequences, rather than the cause, of neuronal dysfunction. The above review of the potential mechanisms underlying the pathogenesis of PD shows that important issues remain 5.2. Protein aggregation and the ubiquitin–proteasome unsolved. First, since 1997, a plethora of genetic defects has system been reported in familial forms of PD, with an autosomal dominant or recessive mode of inheritance. To date, nearly Aggregation of soluble proteins into insoluble complexes and twenty loci have been implicated, from which ten different inclusions is now considered at the core of many neurode- causal as well as susceptibility gene mutations have been generative conditions, including PD. One pivotal mechanism formally identified (Table 2). In general, with the notable for proteins to aggregate relies on the exposure of hydrophobic exception of PARK9, PARK14 and PARK15, the phenotype of segments that are prone to form inappropriate intermolecular genetically-determined PD is quite similar to sporadic PD, but binding, when proteins are unfolded, partially folded or age at onset is usually younger. The involvement of some of misfolded. It is noteworthy, however, that protein aggregation these genes tends to support the mechanisms of degeneration does not seem to occur at random but is a highly specific described above. For example, PARK2 is characterized by genetic phenomenon occurring between similar hydrophobic proteins defects in the protein parkin which acts mainly as an E3 or distinct proteins sharing a common aggregation-promoting ubiquitin ligase essential for the UPS. Similarly, PARK6 results motif [58]. During this process, many native proteins not from mutated forms of PTEN-induced kinase 1, a mitochondrial primarily involved in the aggregation formation, may be protein that seems to have a protective role against proteasome recruited and trapped into growing inclusions, as ”innocent inhibitors. On the other hand, pathogenic mechanisms asso- by-standers”. ciated with PARK8 and more recent gene abnormalities fit less Under physiological conditions, neurons handle abnormal easily into current theories. Although information obtained or altered (genetically modified, denatured, misfolded, etc) through genetic approaches has proved immensely useful to cellular proteins by distinct mechanisms, notably molecular elaborate new hypotheses regarding the pathogenesis of chaperone-mediated refolding, degradation by the ubiquitin– sporadic PD, the diversity of genes uncovered so far makes it proteasome system (UPS) and peptidases, and, when the difficult to establish a unifying hypothesis that takes all or most production of abnormal polypeptides exceeds the capacity of known gene products into account. Second, it is still not clear the UPS, sequestration into microtubule-dependent aggre- whether LBs and LNs are the toxic entities promoting 16

Table 2 – PARK-assigned causal and susceptibility genetic defects leading or contributing to Parkinson's disease. Locus a Gene Mutation Mode of Gene Protein Age of Features Lewy location a symbol a inheritance product function onset bodies

PARK1 4q21.3-q22 SNCA A53T, A30P, E46K AD (sometimes in α-synuclein Synaptic protein of unknown 45- Early-onset PD with Yes sporadic cases) function 60 years early dementia PARK2 6q25.2-q27 PARK2 Point mutations, AR (rarely AD) Parkin E3 ubiquitin ligase <20 years Early-onset PD with A few deletions, multiplications slow progression PARK3 2p13 PARK3 ? AD ? ? 60 years Late-onset typical PD Yes

PARK4 4q21.3-q22 SNCA Duplication, triplication AD α-synuclein Synaptic protein of 45– Early-onset PD with Yes 10 (2009) 73 PROTEOMICS OF JOURNAL unknown function 60 years early dementia PARK5 4p13 UCHL1 Mutation AD Ubiquitin carboxylterminal Deubiquitylating 45– Early-onset typical PD ? hydrolase L1 enzyme 60 years PARK6 1p36.12 PINK1 Missense mutations, AR (rarely AD) PTEN-induced kinase 1 Mitochondrial protein 20– Early-onset PD with Yes truncations of unknown function 45 years slow progression PARK7 1p36 DJ-1 Deletions, truncation, AR DJ-1 protein Astrocytic and neuronal 20– Early-onset PD with ? missense mutation protein of unknwon function 45 years slow progression PARK8 12q12 LRKK2 G2019S, I2020T, AD (sometimes in Leucine-rich repeat / Protein kinase >50 years Late-onset typical PD Usually missense mutations sporadic cases) threonine protein kinase 2 yes PARK9 1p36 ATP13A2 Missense mutations AR ATPase type 13A2 Lysosomal type 5 Ptype <20 years Atypical ? ATPase (Kufor–Rakeb disease) PARK10 1p32 PARK10 ? Susceptibility ? ? 60– Typical PD ? 70 years PARK11 2q37.1 GIGYF2 Missense mutations AD GRB10-interacting GYF protein Brain protein of unknown 60– Typical PD ? 2 function 70 years

PARK12 Xq21-q25 PARK12 ? Susceptibility ? ? 60– Typical PD – 29 70 years PARK13 2p13.1 HTRA2 Missense mutations, Susceptibility Omi/HtrA serine peptidase 2 Mitochondrial serine 60– Typical PD ? polymorphism protease 70 years PARK14 22q13.1 PLA2G6 Missense mutations AR Phospholipase A2, group VI Fatty acid release from 10– Atypical ? phosphlipids 25 years (dystoniaparkinsonism) PARK15 22q11.2-qter FBX07 Missense, truncating, AR F-box protein 7 Molecular scaffold in the 10– Atypical ? compound mutations formation of protein complexes 20 years (pallidopyramidal disease) a According to www.genenames.org (March 2009). JOURNAL OF PROTEOMICS 73 (2009) 10– 29 17 degeneration by impairing the normal functioning of neurons degeneration of DA neurons and to establish the basis for future and axons, or whether it represents a protective mechanism of neuroprotective therapies, they also demonstrated a number of defence by which neurons sequester altered proteins to limit limitations. At present, none of them consistently recapitulate their toxic effects upon neuronal homeostasis [66]. In the latter all clinical and neuropathological features of human sporadic scenario, degeneration occurs late in the process, when the PD. Moreover, it has been particularly difficult to model the capacity of LBs to circumvent toxic compounds is overwhelmed. progressive time course of PD, to replicate the pattern of A major argument in favor of a protecting role of LBs in PD comes neurodegeneration involving selected nigral and extra-nigral from the observation that PARK2- or PARK8-affected individuals brain structures over time and to produce typical LBs [73]. While do not consistently show LBs, suggesting that LBs are not almost each group working on PD basic research has been able necessary for nigral degeneration to occur. The neurotoxicity of to produce its own animal model of PD, the gold standard in this elevated levels of various α-SYN intermediates in the aggrega- field is still awaited. Proteomics technologies have been applied tion process (monomers, oligomers, protofibrils and fibrils) has to some of these animal models [74–87], and these aspects — also been extensively studied (recently reviewed in [67–69])and which extend beyond the scope of the present article devoted to each has been suspected to be the triggering agent of degenera- human sporadic PD — have been extensively reviewed in [88] to tion in PD, rather than mature LBs. This issue is crucial with which the reader is referred for more detailed information. respect to developing neuroprotective agents, because inter- ventions aiming at interfering with protein aggregation may 6.2. Proteomics in human PD turn out to be deleterious by inhibiting the formation of potentially protective LBs or by favoring the accumulation of In contrast to animal models which can be quite easily made toxic intermediates. Third, the temporal and mechanistical available as needed, the type, number, quantity and quality of relationships between oxidative stress, mitochondrial dysfunc- samples that can be obtained from humans are limited. tion and protein aggregation are still unclear and, perhaps more However, samples from neurologically-affected patients are importantly, the promoters, i.e. the causal factors, of these of enormous interest because they may show abnormalities abnormal biological processes are completely unknown [70,71]. directly linked to the condition under study. In PD, as in other Fourth, the selectivity of the structures involved in PD, as neurological diseases, most studies have relied on samples of described above, can hardly be explained by the currently cerebrospinal fluid (CSF) obtained from living patients and of known pathogenic factors likely to operate in the PD brains, and brain tissue obtained at autopsy, the value of each being the morphological hypotheses developed by Braak and collea- dependent upon the proposed aims of the study. Both types of gues are difficult to incorporate into the suspected scenarios sample have advantages and limitations that require to be associating oxidative stress, mitochondrial dysfunction and recognized when interpreting data. proteasome dysfunction. In order to examine these unanswered, yet fundamental 6.2.1. CSF protein profiling questions, and because PD is now viewed as a disorder of brain CSF may represent an ideal source of diagnostic, prognostic of protein handling — or a “protein deposition/conformational therapeutic biomarkers, because of its proximity to brain disease”—we make the assumption that proteomics-based structures undergoing degeneration which may release patho- studies of relevant brain samples might be one of the most appro- genically relevant molecules into the extracellular space. In priate and unbiased strategies to approach PD pathogenesis. addition, CSF is available over patient's lifetime which makes possible the study of protein profile changes during the entire disease course. Indeed, following the introduction and routine 6. Proteomics applied to Parkinson's use of atraumatic needles in the clinic, lumbar puncture (LP) is disease research now considered a minimally invasive procedure that can be performed at any time during PD progression and repeated in During the past few years, a number of proteomic studies have the same individual. The quantity of CSF that can be obtained been performed in the field of PD research both in animal from a single LP is between 5 and 20 ml, which corresponds models of and in humans with PD. roughly to 2 to 8 mg of proteins, a figure that allows most proteomic technologies to be performed in multiplicates. 6.1. Proteomics in animal models of PD Proteomic approaches of CSF have already proved successful for other neurodegenerative conditions such as AD [89] and Numerous animal models of PD have been designed using Creutzfeldt–Jakob disease [90]. Extensive CSF 2-DE maps have species as diversified as the worm Caenorhabditis elegans, the fly been published [91,92] as well as using other separation methods Drosophila melanogaster, various strains of mice and rats, and [93,94] and, thus far, over 2500 proteins have been identified in non-human primates [72]. Neurodegeneration has been human CSF [95]. Advantages and limitations regarding the use of obtained by means of various protocols of exposure to the CSF in proteomics have been discussed elsewhere, in particular neurotoxins 6-hydroxydopamine, MPTP, paraquat, rotenone or with respect to sample preparation which requires specific to proinflammatory molecules such as lipopolysaccharides. handling [96,97]. One of the major issues relies on the high More recently, genetically-modified animals have been engi- dynamic range of protein concentrations contained in CSF, a neered according to the identified gene defects found in small number of which overwhelming a multitude of low humans. While these animal models have proved very useful abundance, yet of key value, proteins. This problem may be to increase our understanding of the pathophysiology of the solved by pre-analytical fractionation or immunoaffinity techni- basal ganglia, to dissect some mechanisms engaged in the ques allowing the few more abundant proteins to be removed. 18 JOURNAL OF PROTEOMICS 73 (2009) 10– 29

In PD, because degeneration affects only a limited subset Currently, no single biomarker has been validated for PD of brain structures, it is anticipated that potential biomarkers and it is possible that a combination of several of them may will be found as low or very low abundance proteins, as prove superior. For example, a large scale study using demonstrated, for example, by the CSF determination of the Luminex-based multianalyte profile reported the performance α β monomeric form of -SYN which has proved difficult because of a panel of eight CSF proteins (tau, amyloid β42, 2- of its minute concentration in this biological fluid. Recently, a microglobulin, vitamin D binding protein, apolipoprotein A-II group firmly established the measurement of α-SYN in plasma and apolipoprotein E, brain derived neutrophic factor, and and CSF [98]. On average, a decreased concentration of α-SYN interleukin-8) to distinguish PD from AD patients and controls. was observed in the CSF of PD patients compared with age- Authors were able to identify effectively PD patients with a matched controls and this decrease correlated with disease 95% sensitivity and 95% specificity [109] and these encoura- severity, although values from both groups were largely ging results will require confirmation by independent groups. overlapping [99]. Moreover, CSF levels of DJ-1 were found to be significantly increased in sporadic PD and well correlated 6.2.2. Brain tissue protein profiling with disease progression [100]. DJ-1 was also found into the At variance with the use of samples obtained from living serum of both healthy controls and PD patients, but no patients, in which only a tentative — probable at the very best — significant level differences were observed between the two diagnosis of PD can be made, brain samples have the consider- groups [101]. The distinct behavior of DJ-1 levels between CSF able advantage of being assessed by neuropathological exam- and serum could be explained either by a diluting effect of the ination, providing a definite diagnosis of PD. Regions of interest protein in serum or by a potential oxidation of the protein can be precisely dissected, for example the SNpc, LC or cerebral that may have prevented antibodies directed against native cortex, thus increasing the study specificity. The large amount DJ-1 to recognize the post-translationnally modified protein. of tissue available may allow individual cells, i.e. DA neurons to The latter hypothesis is supported by the fact that abnor- be separated, or subcellular fractions, i.e. LBs, to be purified. mal oxidation of DJ-1 has indeed been observed in PD patients Finally, and more importantly, direct analyses of the very [102]. structures that are primarily affected in PD may provide a Historically, in 1984, Harrington and Merril studied CSF comprehensive and simultaneous view of the degenerative samples from 20 PD patients by the then available 2-DE mechanisms that are likely to be at work. The major theoretical technology and were able to detect in 75% of their cases, but limitation of this approach involves the potential contamina- in none of 91 healthy controls, the appearance of a single tion of analyses by post-mortem alterations [110]. In fact, this abnormal spot (129 in their nomenclature) present within the issue has been recently examined by independent groups using Ig light chains train of spots [103]. Quantitative changes of 20 human [111] and mouse [110, 112, 113] brain tissue samples additional spots were also noted, notably apolipoprotein A1 exposed to increasingly long post-mortem interval, up to 72 h. and orosomucoid. Unfortunately, these findings were never Somewhat surprisingly, these studies consistently showed that, confirmed by this group nor replicated by others ever since. even for prolonged post-mortem delay at room temperature, More recently, a more advanced study based on a multiplex brain tissue does not undergo massive degradation and only a quantitative platform using iTRAQ was able to detect various minority (6.5% of spots after 48 h at room temperature in [111])of changes in the composition of CSF from patients with AD, PD, proteins show significant modifications, including dihydropyr- dementia with LBs and controls [104]. Over 1500 proteins were imidinase related protein-2, GFAP and, importantly for PD identified and 72 were reported to be uniquely associated with research, synuclein proteins. Most changes occur after 48 h. PD. Ceruloplasmin and apolipoprotein H were confirmed by The problem of artifactual post-mortem changes may thus not Western blot analysis and appeared to be good candidate be as significant as previously feared and can be circumvented markers for PD. For instance, the deposition of ceruloplasmin, by limiting the post-mortem delay to less than 24 h and by a major iron-carrying protein, had already been found to including in the study a control group of neuropathologically correlate with disease severity [105] and to be decreased in the intact brain samples with matched post-mortem delay, when- blood of PD patients [106]. However, a recent study challenged ever possible. Lists of proteins vulnerable to post-mortem delay the significance of this finding, as considerable overlap in are available in [110] and [111]. Additional issues include the individual values between PD patients and controls was contribution of unspecific age-related changes to the pathology, observed [107]. In another study, the sera of 422 patients with the potential impact of the many and long-used pharmacolo- PD, amyotrophic lateral sclerosis (ALS), related neurological gical treatments upon protein expression and a selection bias disorders and unaffected individuals were investigated using owing to the fact that, with few exceptions, only patients with quantitative 2-DE [108]. In this unbiased approach, differences advanced PD come to autopsy. This last point is important in the expression of 7 out of 9 complement-related proteins because it may obscure data interpretation especially when were found. The PD and ALS populations showed a marked assessing changes of protein expression for the purpose of increase in components of complement C3 (C3c and C3dg) and establishing early biomarkers of the condition. Ideally, PD complement factor H. Moreover, factor B was more elevated in patients with large range of disease durations should be studied. PD, but not in ALS, with respect to controls. These results Fasano et al. were the first to profile the human PD SNpc showing differences in the pathways of complement, includ- using 2-DE. This group was able to identify a total of 44 proteins ing C3 phosphorylation differences, between ALS and PD from which nine were found differentially expressed in PD provide independent evidence for the role of systemic, compared to age-matched controls [114]. Of note, there was an immune-mediated inflammatory processes possibly involved increase of some ROS-scavenging and mitochondrial proteins in these conditions. (peroxiredoxin II, mitochondrial complex III or ATP synthase D) JOURNAL OF PROTEOMICS 73 (2009) 10– 29 19 in PD specimens, supporting the role of oxidative stress in PD systems, such as PCR for nucleic acid, as well as the time pathogenesis. Using a similar approach, the SNpc study by consuming and cumbersome nature of cell collection. Indeed, Werner et al. led to the detection of 221 spots found differen- the large number of cells to be extracted from a tissue section tially expressed between PD and control groups, contrasting necessary for 2-DE analyses [123–125] or a shotgun proteomics with 321 spots showing a strictly conserved pattern of expres- analysis followed by MS [126,127] may vary from 10,000 to sion. Out of a total of 37 identified proteins, they found 16 100,000 cells. These figures may decrease in the near future differentially regulated, including elements of iron metabolism with the emergence of more sensitive MS techniques and (ferritin H), glutathione-related redox metabolism (glutathione automated methods to collect cells. Other purification meth- S-transferase M3, P1, O1) or novel redox proteins such as ods have been tested for this purpose but it is uncertain SH3BGRL [115]. Importantly, expression of DJ-1 and UCH-L1 whether these techniques are suitable for proteomics [128– was not different in the two groups and twelve of the 16 130]. Leverenz et al. examined the content of LBs dissected by differentially regulated proteins displayed only modest LCM from neurons of the frontal cortex from patients with changes, as the level ratio of the PD over control group was dementia with LBs [131]. The analysis of approximately 2500 below 1.5 fold. Comparing the data from these two similar cortical LBs led to the identification of 296 proteins, of which 17 studies, 8 common proteins were found yet none of the had been previously associated with brainstem or cortical LBs, differentially expressed ones were concordant. More recently, including α-SYN. The presence of one protein, the heat shock an extensive investigation of the human SNpc using a shotgun cognate 71 was validated by immunohistochemistry not only proteomic approach combining ICAT or iTRAQ and MS or MS/MS in the brainstem but also in cortical LBs. More recently, another identified a total of 1263 non-redundant proteins, but the group, using LCMS/MS, studied cortical LBs from patients with complete dataset of protein identifications was not provided the Lewy variant of AD that were enriched by sucrose gradient [116]. centrifugation [132]. These researchers were able to identify As LB pathology eventually extends from the brainstem to about 550 proteins from which 40 were found to co-purify with the cortex, efforts were also made to characterize the human LBs including some well known LB-associated proteins such as frontal cortex in PD [117]. Using LCMS/MS-based technology α-SYN, UB, UCH-L1 and proteasomal components. Interest- and iTRAQ labelling, a study reported the characterization of ingly, they also identified novel kinases, the deubiquitinating human cortical proteins affected during PD progression [118]. enzyme otubain 1 and UB ligases (KPC, skip-cullin-F-box and About 800 proteins were identified, and approximately 200 possibly Ube4b). Similarly to proteomic studies of the SNpc, were found to be differentially expressed between PD patients comparison of results between both studies on cortical LBs at various stages and controls, including the 70-kDa mito- showed minimal concordance, raising the possibility of biases chondrial heat shock protein mortalin which was found to related to samples or methods differences. decrease with disease progression. Recently, it has been Because the physiological functions of proteins are likely demonstrated that CRP40, a splice variant of mortalin, exhibits related to their specific intracellular localization, isolation of a catecholamine binding function that may confer neuropro- these specific cellular compartments can be considered as an tective properties to this chaperone-like protein [119]. entry point to understand the cellular mechanisms underlying brain disorders. Organelle proteomics consists in the enrich- 6.2.3. Cellular and subcellular proteomes ment and isolation of fractions containing proteins associated To allow a deeper probing of the neural proteome, various with organelles of interest via classical methods such as strategies have to be elaborated to reduce the complexity of the fractionation, immunoprecipitation or affinity pull-down stra- sample prior to analysis and to select only a discrete, but highly tegies, among others, prior to proteomic investigation (reviewed relevant subset of the entire proteome [120]. When using PD in [133–135]). Various subcellular structures such as mitochon- brain samples, this concept translates into assessing affected dria [136,137], plasma membranes [138], synaptic vesicles [139] tissue and cells with an increasing level of specificity, ranging or post-synaptic structures [140,141] have been already studied from the entire SNpc, or other LBs-enriched regions, to DA using proteomic approaches, and may contribute to understand neurons and eventually to LBs and other relevant subcellular their role in PD pathogenesis. For example, extensive investiga- or even molecular fractions. tions identified and quantified proteins of mitochondria- It is important to appreciate that the SNpc is composed of enriched fractions isolated from human SNpc of MPTP-treated different cell types among which only 5 to10% are DA neurons. mice and PD patients compared to age-matched controls using Thus, a 5-fold change in protein expression in DA neurons will an unbiased quantitative proteomic approach [77,142].Inthe be diluted to 0.5-fold or less and hardly detectable when latter, by comparing the two groups differentially labelled with examining the whole SNpc. Recent improvements in sampling ICAT and analysed by a linear trap quadrupole (LTQ) mass techniques such as the development of laser capture micro- spectrometer, authors reported the identification of as much as dissection (LCM) have allowed the separation of cell popula- 842 proteins with 119 displaying significant changes in their tions of enhanced purity from heterogeneous tissue sections relative abundance. Interestingly, mortalin was found again to be via direct visualization of the cells of interest [121]. With this substantially and preferentially decreased in the mitochondrial technique, a finely-focused laser is used to cut around or fractionsofPDbrainsaswellasintherotenone-inducedcell capture cells of interest out of fixed tissue slices (reviewed in model of PD. Another study reported a sequential method for the [122]). Although LCM was commonly applied in genomic isolation of highly purified NM granules found in DA neurons of molecular profiling, the approach remains limited in proteo- human SNpc prior to proteomic analysis [143]. Seventy-two mics. The main drawback, precluding a more widespread proteins were identified by LCMS/MS analysis, of which the proteomic application, lies in the lack of protein amplification majority was shared with melanosomes and lysosomes. These 20 JOURNAL OF PROTEOMICS 73 (2009) 10– 29 results provide important insights into the cellular processes which comprise around 30% of eukaryotic proteins [148] and are generating the NM granules. As many evidences suggest a naturally prone to aggregation. To verify this hypothesis, a functional role for NM in PD neurodegeneration [21,22,144,145], screening of the primary sequences of the identified proteins may the comparison of NM granules content between control and be undertaken using algorithms that have been developed to diseased states may offer some important clues regarding NM predict these regions of extended disorders [149].Finally,α-SYN toxicity in PD. was shown to display a large number of binding partners in cell Finally, a body of evidence suggests that PTMs may contribute lines [150].Ifα-SYN itself is not toxic when present at to disease pathology, particularly in the SNpc. Approaches physiological concentrations, its interaction with other mole- allowing the high throughput proteomic analysis of specifically cules could lead to neuronal degeneration and, again, the modified subproteomes such as oxidised, phosphorylated, proteomic characterization of these interactants by pull-down ubiquitinated or glycosylated [146] proteins have been developed. assays on LB from post-mortem tissues may be of interest. Until now they were mainly applied to cell lines or animal models Consequently, we propose a reappraisal of the protein and to a lesser extent to human brain tissues of AD or PD patients. aggregation hypothesis that, in our opinion, should go beyond the study of α-SYN alone. We here hypothesize that PD may be a more widespread brain “proteinopathy” (or protein conforma- 7. Potential avenues and conclusion tional disorder) whereby a number of aggregation-prone neuronal proteins may experience conformational modifica- After more than a decade of intensive research on α-SYN tions that promote abnormal protein-to-protein interactions aggregation, it becomes increasingly clear that α-SYN cannot by and increase binding capacity between related but not necessa- itself elucidate all aspects of PD pathogenesis. First, as already rily similar, hydrophobic, natively unfolded or partially folded mentioned above, α-SYN aggregates are not unique to PD but can polypeptides. These conformational modifications may result be found in a variety of conditions grouped under the label from distinct mechanisms, including overexpression, PTMs or synucleinopathies, a group of enormously heterogeneous condi- miscleavage of one or a few aggregation-prone proteins, and tions with respect to their clinical manifestations, neuroradiolo- may initiate a cascade of events by which these proteins may gical expression and neuropathological features. α-SYN can also become inactive or toxic. Then, small aggregates formed any- be found incidentally in otherwise neurologically intact indivi- where within neuron body and processes may coalesce and duals. Even at the cellular level, α-SYN deposition may occur not grow into insoluble, degradation-resistant inclusions. Identify- only in neurons but also in other cell types, such as olidoden- ing which proteins are best candidates for these events to occur drocytes in multiple system atrophy where they are referred to as and deciphering which mechanisms are more likely to be glial cytoplasmic inclusions. Second, α-SYN pattern of immu- involved, are all crucial issues that may be addressed by means nostaining is primarily located in the periphery of mesencephalic of specific pre-analytical and analytical proteomic strategies. LBs and is not prominent in its core as would be expected for a To achieve this goal, we believe that much effort should be molecule presumed to play an initiating role in seeding aggrega- placed on sample purification and fractionation prior to tion. Conversely, ubiquitinated proteins and components of the proteomic analyses. For the study of human SNpc, as shown UPS are found in the LB centre. Third, based on its intense on Fig. 2, we propose an experimental workflow that allows staining using immunohistochemistry techniques, α-SYN is isolating proteomes of interest with an increasing level of considered a major if not the essential component of LBs. tissular, cellular and subcellular specificity. At the tissular level, However, immunostaining methods are notoriously not quanti- the mesencephalon from neuropathologically-proven PD cases tative and, in fact, a truly quantitative assessment of the many LB and matched controls should be carefully and selectively components is still awaited. Furthermore, the totality of LB dissected, frozen and cryosectioned. Delineating the whole constituents has not been established yet and proteomic studies SNpc proteome and comparing it between the two groups may on cortical LBs [131,132] suggest that α-SYN is just one among provide information on global changes occurring in this hundreds of proteins that compose LBs. Fourth, while numerous structure in PD. At the cellular level, a proteomics-compatible studies have focused on α-SYN and its high propensity to number of bromophenol blue stained-DA neurons are extracted aggregate under certain conditions, limited information is from the same sets of samples using LCM, yielding three dis- available about other proteins that may exhibit similar properties tinct neuronal populations to be compared, those from healthy suchasparkin,UCH-L1,LRKK2,DJ-1,omi/HtrA2,PINK1and subjects and those from PD neurons with and without LBs. others. For example, the in vitro overexpression of parkin Rather than focusing on LBs, this step may be important associated with an inhibition of proteasomal activity, was to assess the whole cellular pathophysiology and particu- recently shown to lead to the formation of inclusions sharing larly to study LB-free nigral degeneration that occur in some many characteristics with LBs such as their organisation or genetically-determined forms of PD such as PARK2. At the immunoreactivity against UB, and some proteasome compo- subcellular level, proteomes of interest from the same sets of nents [147], supporting the notion that other proteins than α-SYN neurons can be determined after enrichment of relevant may induce aggregation. A way to identify aggregation-prone fractions, including nuclei, mitochondria, synapses, NM gran- proteins may rely on their primary aminoacid sequence, as ules, LBs or other complexes, through subcellular fractionation proteins containing the hydrophobic aminoacids Ala, Val, Leu, Ile, or immunoprecipitation techniques. It is noteworthy that LCM Phe, Trp, Met or Pro may be more at risk of misfolding or being may not be suitable to purify LBs because of their small size resistant to refolding. It is well possible that many LB constituents which approaches the capture limits of the LCM technology belong to this class of hydrophobic proteins. In addition, LBs may [132]. Because the same samples are used at each step, be enriched with natively unfolded proteins, such as α-SYN, comparison of datasets may be less biased by inter-individual JOURNAL OF PROTEOMICS 73 (2009) 10– 29 21

Fig. 2 – Diagram showing a possible proteomic approach to assess biological mechanisms underlying neurodegeneration in the substantia nigra pars compacta from Parkinson's disease human brains obtained at autopsy. At the pre-analytical level, it is proposed to examine samples with an increasing level of specificity at each step and with appropriate proteomics strategies, according to working hypotheses.

variability. Specific proteomic strategies are defined for each comparison of silver-stained patterns of multiple gels and MS/ level, according to working hypotheses, which involve (1) estab- MS identification remains valid, the development of fluorescent lishing which proteins may be over- or underexpressed in the and chemiluminescent based technologies has extended its SNpc and its components, using quantitative approaches; application, allowing the multiplex assessment of protein (2) examining which qualitative protein modifications may play quantification on a single gel using the DIGE technology and a prominent role in the aggregation processes; and (3) assessing the large scale detection of protein modifications. MS/MS protein miscleavage as another potential mechanism under- identification should be performed with highly sensitive mass lying inclusion formation in PD. Data validation is a crucial step spectrometers that may be able to dissect spots into their that should not be forgotten along the whole procedure. sometimes multiple components. Another method called “shot- gun proteomics” involves proteins trypsinisation, peptide 7.1. Quantitating protein expression separations using a broad range of multi-dimensional chroma- tographic, affinity or gel-based techniques, followed by MS/MS Multiplication of the SNCA gene has been shown to be a identification. This gel-free alternative has become widespread potential mechanism by which increased expression of wild- for relative or absolute quantification using stable isotope labels type α-SYN may lead not only to autosomal dominant but also at a protein or peptide level, and more recently for label-free to apparently sporadic PD with LBs. Interestingly, phenotype quantitation strategies. In stable isotope labelling, quantitative and disease severity seem correlated with SNCA copy number peptide ratios — thought to reflect accurately the abundance and probably gene product levels [151]. This observation ratio of the compared proteins — can be obtained from the provides the rationale to study other proteins, whose over- relative comparison of peptide signal intensities discriminated expression and high intracellular concentrations may promote from single MS or MS/MS scans owing to their mass differences inclusion formation. Quantitative or semi-quantitative meth- (for reviewed see [152,153]). For example, the isobaric tagging of ods are highly desirable to examine this hypothesis. Whereas peptides using iTRAQ or tandem mass tags (TMT) [154] the traditional proteomic approach using 2-DE separation, consisting in a reporter and a balance group, enables the 22 JOURNAL OF PROTEOMICS 73 (2009) 10– 29 simultaneous protein identification and quantification of up to the melanised neurons of PD brain [165]. The 2DOxyblot respectively 6 and 8 samples by MS/MS. After samples pooling, technique was also successfully used to map nitrated proteins peptides of the same sequence labelled with different versions in AD brains [166]. It is therefore anticipated that the application of a set of multiplex isobaric reagents appear at the same mass of 2DOxyblot on human SNpc, DA neurons or subcellular in an MS scan, but upon CID fragmentation in the mass fractions could provide new insights into PD mechanisms. spectrometer, give rise to low mass MS/MS signature ions A third approach has recently emerged from the demonstra- corresponding to the reporter loss. In addition, when proteins of tion that the nitrosylation reaction of cysteine thiol side chain interest are identified, the spiking of a known quantity of an with nitric oxide species forms S-nitrosylated derivatives. This isotope-labelled peptide chemically synthesized as an internal mechanism has recently been linked to unfolded protein standard can provide absolute quantification [155].Incontrast, response [167] and neurodegeneration in PD [167–169]. The label-free quantitation can either be based on the correlation of detection of endogenously S-nitrosylated proteins mainly relies the MS peak intensities with protein amounts [156] or on the on the biotin-switch assay pioneered by Jaffrey and Snyder and is correlation of the number of MS/MS spectra assigned to each compatible with a broad range of methods such as SDS-PAGE, protein (spectral counting) [157]. immunodetection and mass spectrometry [170]. Using this All these quantitative approaches can be used at any step in technique, the protein–disulphide isomerase (PDI) chaperone, the workflow proposed on Fig. 2, although we believe gel-based which assists in the maturation and transport of unfolded techniques may be insufficiently sensitive and therefore less secretory proteins was shown to be S-nitrosylated in PD brains. suitable for cellular of subcellular fraction analyses. To make The modification blocked its protective enzymatic activity, results more robust, we also propose to use two independent leading to the accumulation of polyubiquitinated proteins and methods of quantification using the same set of samples, in activating the unfolded protein response. These data support a order to identify — and exclude — method-dependent data, previously unrecognized relationship between nitric oxide and and to apply stringent criteria for the determination of protein misfolding in degenerative disorders [167].Moreover,two differentially expressed spots or MS peaks. studies reported that parkin was S-nitrosylated in various cellular andanimalmodelsaswellasinsporadicPDbrains[168,171]. 7.2. Examining protein modifications These authors found that, although overexpression of parkin could rescue neuronal cell lines exposed to proteasome inhibi- Proteomic tools allow performing comprehensive study of tion, the S-nitrosylation could inhibit parkin ubiquitin E3 ligase specific PTMs that are known to occur in the PD SNpc, activity and alter its protective function [168,171]. S-nitrosylation including the identification and quantification of modified may preclude proper ubiquitination of parkin substrates resulting proteins as well as the precise sites of modification. in their aggregation. These findings provide a molecular link between nitric oxide toxicity, UPS impairment and protein 7.2.1. Oxidation, nitration and S-nitrosylation accumulation in sporadic PD. In PD, considerable evidence supports oxidative or nitrative Lately, a high throughput proteomic approach termed stress as key factors involved in neurodegeneration [158]. For SNOSID (S-NitrOsylation Site Identification) has been devel- example, oxidative modifications of α-SYN via dopamine oped for the simultaneous identification of proteins contain- adducts [159] or nitration [160] facilitate its aggregation. ing SNO-cysteine sites in complex biological mixtures [172]. Three assays are commonly used to assess markers of protein Built on the biotin-switch technique, this innovative method oxidation based on the detection of protein carbonyls, combines the biotinylation of protein SNO-cysteine residues nitration or cysteine S-nitrosylation. followed by trypsin digestion, affinity purification of biotiny- The first assay involves the reaction of 2,4-dinitrophenyl- lated-peptides, and amino acid sequencing by liquid chroma- hydrazine with carbonyl groups on proteins, followed by their tography tandem MS [172]. To the best of our knowledge, this spectrophotometrical quantification. This technique was applied approach has not yet been used in PD research. to SNpc specimens from PD patients and demonstrated increased levels of oxidised proteins compared to controls [161,162]. The 7.2.2. Phosphorylation high throughput comparison and identification of proteins Protein phosphorylation is one of the most frequent PTMs and a containing carbonyls in diseased and control brain samples can critical regulatory mechanism of cellular homeostasis including be determined by an elegant method termed 2DOxyblot, without proliferation, gene expression or signal transduction [173–175]. loosing information on protein isoforms. The technique couples For example, in AD and other tauopathies, a massive increase in 2-DE with the immunohistochemical detection of protein reactive the concentration of free hyperphosphorylated tau is thought to carbonyls groups and finally the matching of 2DOxyblot with 2-DE be at the of abnormal filaments assembly. In synucleino- gels images followed by mass spectrometry of differentially pathies, α-SYN was found to be extensively and selectively detected spots. This procedure was successfully applied to the phosphorylated at Ser129 in aggregates, as demonstrated by MS comparison of oxidation levels of brain proteins in the inferior and histochemical techniques [176], and this modification parietal lobule of AD subjects versus controls, with the identifica- seems to promote fibrillogenesis in vitro [176]. tion of several specifically oxidised proteins, such as creatine A variety of strategies have been introduced for the large kinase BB, glutamine synthase, UCH-L1 and others [163,164]. scale identification of phospho-proteins and phospho-peptides, A second way to index oxidative damage to proteins is the determination of their sites of phosphorylation on Ser, through the nitration of tyrosine residues mediated by reactive Thr or Tyr residues and their quantitative analysis (see review nitrogen species. Antibodies specific for 3-nitrotyrosine were [177–179]). Quantitative phosphoproteomics is based on 2-DE or used to demonstrate increased protein nitrations within LBs in MS technologies. The use of phosphorylation-specific stains JOURNAL OF PROTEOMICS 73 (2009) 10– 29 23

Table 3 – Some proteomics-derived proteins of interest for PD research. SwissProt Protein name Function Sample Quantification Observed Hypothetic Reference ID method changes a significance

P00450 Ceruloplasmin Iron transporter CSF iTRAQ LCMS/MS ↓ (0.65) Free iron- [101] induced oxidative stress P02749 Apolipoprotein Transporter of various substrates CSF iTRAQ LCMS/MS ↓ (0.66) Unclear [101] H (i.e. phospholipids, heparin) P01024 Complement Serum 2DE ↑ (~7) [105] proteins C3 isoform C3c phosphorylated P01024 Complement Immune/inflammatory response Serum 2DE ↑ (~5.5) proteins C3 through complement system isoform C3dg activation P08603 Complement Serum 2DE ↑ (~3) [105] factor H, short spliced form P54289 Voltage- Calcium-dependent signal SNpc 2DE ↑ (2.7) Increased [108] dependent transduction calcium influx in calcium DA neurons channel subunit alpha- 2/delta-1 P32119 Peroxiredoxin 2 Redox metabolism SNpc 2DE ↑ (2.0) Acts against [108] increased oxidative stress O75368 SH3 domain Redox metabolism SNpc 2DE ↑ (1.85) Acts against [109] binding increased glutamic acid oxidative stress rich protein like (SH3BGRL) O75947 ATP synthase D Energy production SNpc 2DE ↑ (1.75) Compensatory [108] chain mechanism for ATP production inhibition P47985 Ubiquinol Energy production SNpc 2DE 2 spots: ↑ Compensatory [108] cytochrome c (3) and ↑ mechanism for reductase iron– (1.6) ATP production sulfur subunit inhibition O95299 NADH- Energy production SNpc, ICAT LCMS/MS ↓ (0.34) Mitochondrial [73] ubiquinone mitochondrial complex I 42 kDa subunit fraction dysfunction P33518 Cytochrome c Energy production SNpc, ICAT LCMS/MS ↓ (0.69) Mitochondrial [73] oxidase mitochondrial complex I polypeptide 1 fraction dysfunction Q14019 Coactosin-like Cell structure, binds to SNpc 2DE ↑ (2.0) Structural [109] 1 F- reorganisation P09455 Cellular Intracellular transport SNpc 2DE ↑ (1.76) Compensatory [109] retinol-binding of retinol and retinal mechanism for protein 1 loss of aldehyde dehydrogenases P00352 Aldehyde Retinoid metabolism, SNpc, ICAT LCMS/MS ↓ (0.38) Decreased [73] dehydrogenase detoxification of aldehydes mitochondrial detoxification of 1A1 fraction aldehydes SNpc 2DE ↓ (0.75) [109] P38646 Mortalin Chaperone-like protein, control SNpc, ICAT LCMS/MS ↓ (0.45) Reduced [73] of cell proliferation, cellular aging mitochondrial capacity to fraction interact with Frontal iTRAQLC/MSMS, ↓ (0.66– multiple binding [112] cortex, 2DE 0.49) partners or to cytosolic refold proteins fractions a In parentheses, PD samples over controls ratio. 24 JOURNAL OF PROTEOMICS 73 (2009) 10– 29 such as Pro-Q Diamond fully compatible with other staining tool in assessing the role of proteolysis in PD neurodegenera- methods (e.g., DIGE) and modern MS-based analysis [180] has tion, as recently shown by Nilsson et al. [200]. allowed parallel determination of protein expression changes As summarized in Table 3, proteomic studies performed and altered phosphorylation patterns within a single 2-DE gel thus far in the field of PD have already generated a large experiment [181]. This labor-intensive approach is now sup- amount of data that support existing pathogenic theories and planted by the development of phospho-proteins/peptides have produced a number of original findings regarding PD enrichment methods combined with elegant MS identification pathogenesis and biomarkers. However, these developments and quantification using stable isotope labelling [182] or label- remain confidential in the neuroscience community and are free methods [183]. rarely incorporated into the current scenarios of molecular mechanisms underlying PD. Reasons may be found in some 7.2.3. Ubiquitination limitations that tend to affect existing clinical (and basic) Covalent addition of single or multiples units of UB, typically to proteomic studies [201], notably (1) the lack of well-defined and lysine residues, is a crucial mechanism involved in the targeting specific working hypothesis preceding the design of the of intracellular proteins for 26S proteoasomal degradation but proteomic protocol, which has been used essentially as a also in various other functions [184]. UB is a small, highly screening tool; (2) the choice of samples which may not be conserved protein that modifies substrates through a cascade adequate for the purpose of the study; (3) an enormous involving activating (E1), conjugating (E2), ligase (E3) and variability of pre-analytical and analytical methods which deubiquitinating enzymes (DUBs) [185]. As human genome may preclude inter-laboratory comparisons; (4) the limited may encode a few E1s but around fifty E2s and hundreds of E3s, number and heterogeneity of samples examined and low the scope of protein ubiquitination under pathological condi- statistical power of comparative analyses; (5) the absence of tions could be large [186]. The study of ubiquitinated proteins result validation using independent, standardized and reliable provides a direct tool to assess the role of the UPS pathway in PD methods. These caveats have to be kept in mind when pathogenesis. If protein aggregation and LB formation do assessing available studies performed in PD research and originate from an impaired 26S proteasomal function [187] or need to be overcome in future proteomic studies. Based on the from some altered DUBs, then the majority of LB components, or literature reviewed above, it could be concluded that the at least proteins composing LB fibrils, are expected to be fully mechanisms at the basis of PD pathogenesis are exceedingly ubiquitinated or, conversely, deubiquitinated by DUBs following complex and our current understanding of them remains aggregation. This does not seem to be the case in PD but this limited. The prevailing theory involves an impairment of issue requires further investigation. The determination of the selected groups of neurons to handle abnormally processed precise form of UB in aggregates and its substrates might help in cellular proteins which deposit as insoluble and toxic aggre- improving our understanding of the process. Strategies have gates, the cause of which being still elusive. Proteomics- been recently developed for the affinity purification of UB- oriented studies published so far tend to support this view and, conjugates, including UB antibodies [188] and UB binding in fact, suggest that proteomics may be one of the most proteins [189]. However, methodologies still need to be refined promising research avenues in the field of PD pathogenesis. to allow the large scale routine analysis of these modifications. Given the numbers of multi-protein complexes involved in the UPS machinery and all the substrates susceptible to be targeted, Acknowledgments large scale proteomic approaches are necessary to fully under- stand the role of ubiquitination in diseases in general, and in PD This work has been made possible through the generosity of the in particular. Memorial A. de Rothschild Foundation, the Edmond J. Safra Philanthropic Foundation, the Gustaaf Hamburger Foundation, 7.3. 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Chapter III

Neuroproteomics and Parkinson’s disease: don’t forget human samples

Until now, the majority of proteomic analyses published in the field of Parkinson’s disease (PD) were performed in cellular or animal models of PD and proved somehow instructive to dissect some specific molecular pathways involved in PD. However, in the absence of any fully satisfying model able to recapitulate all PD features, results obtained from these studies may be cautiously translated to the human condition. In this editorial, we propose to reappraise the validity of human samples in PD research, which may offer in our view a unique opportunity to detect PD-specific pathological abnormalities. Consequently, we chose to investigate human post-mortem substantia nigra samples and cerebrospinal fluid (CSF) in this thesis project.

I entirely wrote this editorial published in 2011 in Expert review of proteomics (IF=3.7).

Editorial For reprint orders, please contact [email protected] Neuroproteomics and Parkinson’s disease: don’t forget human samples

Expert Rev. Proteomics 8(3), 291–294 (2011)

...we believe that neuroproteomics has an underestimated potential Virginie Licker “ to offer new, unbiased and highly sensitive strategies to study the Neuroproteomics Group, University Medical Center, biological and molecular mechanisms underlying Parkinson’s disease Faculty of Medicine, pathology and to identify diagnostic, prognostic or University of Geneva, Geneva, Switzerland therapeutic biomarkers.”

Parkinson’s disease (PD) is an enigmatic exhibit Lewy pathology, the histological condition, whose clinical manifestations hallmark of PD resulting from the abnormal Pierre R Burkhard and pathological lesions extend far beyond aggregation of a-synuclein (a-SYN) and Author for correspondence the classical tremor, rigidity and akinesia other cellular proteins. The pathology gains Neuroproteomics Group, developed as a result of the degeneration access to the CNS via the olfactory bulbs University Medical Center, Faculty of Medicine, of the dopamine-producing neurons in the and the enteric nervous system, to sequen- University of Geneva substantia nigra pars compacta (SNpc) [1]. tially and selectively target structures of the and Years, if not decades, before motor abnor- brainstem, the limbic system and eventu- Department of Neurology, Geneva University Hospitals, malities start, patients may complain of a ally the cerebral cortex, in a cell-to-cell Geneva, Switzerland variety of nonmotor symptoms including mode of propagation that has been viewed Tel.: +41 223 728 309 loss of sense of smell, sleep disturbances, by some as prion-like [4]. Furthermore, the Fax: +41 223 728 332 [email protected] depression, pain and weight loss among dopaminergic neurons in the SNpc exhibit many others [2], coalescing variably in massive degeneration, whereas adjacent cell individual patients to produce a highly populations are relatively spared. Their spe- heterogeneous phenotype. In addition, as cific vulnerability may rely on their intrinsic the disease progresses, other features may properties as these cells are high-energy con- develop, such as gait disorders, autonomic sumers, contain neuromelanin and elevated dysfunction and cognitive decline, further amounts of reactive species arising from adding to the complexity of the clinical pic- oxygen, dopamine, iron or Ca+2 metabo- ture [2]. As a result, the clinical diagnosis of lism [5]. Finally, PD pathology is not limited PD, mainly based on physical examination to dopaminergic neurons and affects other and some neuroimaging tools, has remained nondopaminergic brain structures well uncertain for a long time, as the distinction before and long after the nigro­striatal sys- from other forms of degenerative parkin- tem [6,7]. Thus, involvement of the SNpc is sonism is difficult, if not impossible, at an just one step in the middle of a much larger early stage. Moreover, patients are necessar- and complex multisystem disorder. ily diagnosed at an advanced pathological The etiology and pathogenesis of PD stage, as the first detectable motor symp- remain equally mysterious. The general toms manifest when approximately 70% scenario suggests that PD is likely to result of nigral neurons are already lost (i.e., long from a subtle interplay involving both a after degenerative processes have been initi- predisposing genetic background and some ated) [3]. Underlying these motor and non- form of environmental toxicity. Indeed, motor manifestations, restricted portions of since the late 1990s, a growing list of muta- the peripheral autonomous nervous system tions in various genes including the a-SYN and CNS selectively undergo neurodegen- gene, SNCA [8,9], have been associated with eration. Vulnerable neuronal populations PD. These mutations mainly account for

Keywords: animal models • biomarker • neurodegeneration • Parkinson’s disease • proteomics • substantia nigra

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early-onset familial forms of PD [10] and those also found in spo- proteomic studies. In the context of PD, we propose proteomics radic cases are very rare. For example, the most common point as an essential exploratory tool that can be used to identify new mutation LRRK2 Gly2019Ser is carried by only 1% of typical, proteins of interest and thus decipher new mechanisms underly- sporadic PD cases and 4% of the hereditary counterpart [11], with ing degeneration, to independently validate existing candidate incomplete penetrance [12] and low concordance in relatives [13]. gene products or hypotheses, and to discover disease biomarkers, The processes by which these mutations lead to Lewy body (LB) another unmet need in PD clinical research. In fact, during the formation and neurodegeneration is unknown [14]. Moreover, a past few years, proteomics has already allowed us to unravel a combination of putative environmental risk factors, such as expo- number of pathways associated with PD patho­genesis as well as sure to various toxins including 1-methyl-4-phenyl-1,2,3,6-tetra­ potential PD biomarkers (for a review see [17]) and we believe that hydropyridine, rotenone or paraquat, and protective factors, such this trend will further increase in the near future. as caffeine consumption, cigarette smoking or anti-inflammatory drug intake, are thought to play a role in PD development [15]. Nonhuman models or human samples? The pathogenesis of sporadic PD is even more elusive despite the A combined PubMed query for ‘Parkinson disease’ and ‘proteome’ numerous mechanisms that have been suggested to account for or ‘proteomics’ terms (up to February 2011) identified 119 papers, neurodegeneration of PD-related structures, including mitochon- including 44 dedicated reviews. Of the 75 original articles, 25% drial dysfunction, oxidative stress, abnormal protein degradation were based on the ana­lysis of human samples (cerebrospinal through the ubiquitin–­proteasome system and/or chaperone- fluid [CSF], blood or brain tissue) whereas the majority (75%) were mediated autophagy, glutamate excitotoxicity, neuro­inflammation, related to cellular and mostly animal models of PD. Various non- increased iron deposition and apoptosis. These mechanisms are human cell lines, invertebrates and rodents were most frequently probably not mutually exclusive and may act at distinct stages of used, whereas one single proteomic study examined a nonhuman PD pathology, with each triggering the next. However, in this view, primate model of PD. Overall, modeling human neurodegenera- it is unclear which pathomechanism is a core element initiating tive diseases has proved to be instructive in understanding their the whole cascade of events and which one is a consequence of it. pathogenesis and has offered platforms to test novel therapeutic interventions. However, disease models have also demonstrated Proteomics as a key tool to study PD serious limitations and, in the field of neurodegeneration, it is pos- At present, PD treatments provide symptomatic benefit at the sible that this traditional approach has reached its limits. PD has expense of invalidating side effects (i.e., dyskinesia) and decreasing been modeled through the administration of neurotoxins in vitro long-term efficacy. PD research is focused on the development of or in vivo or, more recently, genetic manipulations. Such models are neuroprotective or neurorestorative strategies that can slow or halt far from ideal as they do not, or only partly, exhibit the progressive, disease progression. The establishment of such disease-­modifying age-dependent pattern of neurodegeneration involving selected strategies mainly depends on our ability to gain a deep understand- nigral and extra-nigral brain structures, the full spectrum of motor ing of the specific mechanisms under­lying neurodegeneration in and nonmotor symptoms of the condition, or the characteristic order to identify therapeutic targets and diagnose PD accurately LB pathology (for a review see [18,19]). Studies on animal models and at an early stage with new specific and sensitive tools, such should therefore be interpreted with caution and potential patho- as biomarkers. logical modifiers and biomarkers must be ultimately validated in human samples. In fact, it is our view that human samples from PD “...patients are necessarily diagnosed at an patients – either biological fluids from living patients (CSF, blood advanced pathological stage, as the first detectable or urine) or neuropathologically assessed brain tissues – remain motor symptoms manifest when approximately inescapable sources of information and may be an avenue of choice 70% of nigral neurons are already lost.” to detect PD-specific abnormalities through proteomic strategies. Samples from living patients are likely to offer immediately test- In view of the currently limited knowledge surrounding PD able diagnosis, prognosis and therapeutic biomarkers. Although pathogenesis and despite the enormous amount of data already such samples are rarely neuropathologically confirmed, idiopathic available on this topic, we believe it is time to revisit the pivotal PD can be accurately diagnosed during patient’s lifetime with a issues previously described using different, unbiased, non-hypoth- positive predictive value of over 95% when assessed by movement esis-driven approaches. An example of such new lines of thinking disorder specialists [20]. The levels of various pathogenic proteins involves the recent achievement of several genome-wide associa- can be easily and repeatedly measured in the CSF, which might tion studies performed in very large populations of PD patients be a particularly suitable source of biomarkers. Indeed, CSF may and controls, which have been able to link a number of loci to contain pathogenically relevant molecules released by brain struc- increased risk for PD, including MAPT, SNCA, HLA-DRB5, tures and allow the study of protein profile changes at any time BST1, GAK, LRRK2, ACMSD, STK39, MCCC1/LAMP3, SYT11 during the entire disease course. As an example, a-SYN, a major and CCDC62/HIP1R [16]. While the significance of most of these component of LB, has been repeatedly found to be decreased in genes in PD pathogenesis remains to be established, findings from the CSF of PD patients compared with age-matched controls [21–23] genome-wide association studies somehow validate a discovery, and Alzheimer’s disease (AD) patients [22,23]. Hong and colleagues hypothesis-free approach, a strategy that is highly typical of most recently demonstrated that both total DJ-1 and a-SYN levels in

292 Expert Rev. Proteomics 8(3), (2011) Neuroproteomics & Parkinson’s disease: don’t forget human samples Editorial human CSF may represent useful PD diagnostic markers, when shotgun approaches combined with mass spectrometry techniques, blood contamination and age factors are controlled [23]. It is also may allow us to dig increasingly deeper into the proteome of PD possible that a combination of several biomarkers may prove supe- relevant structures. rior to individual ones. For example, a large-scale study reported Irrespective of the samples tested, patient selection and sample the performance of a panel of eight CSF proteins (tau, amyloid quality are of enormous importance for proteomic approaches.

β42, β2-microglobulin, vitamin D-binding protein, apolipoprotein Patients from the ‘Parkinson’ and ‘control’ groups should be carefully A-II, apolipoprotein E, brain-derived neutrophic factor and IL-8) selected and PD diagnosis of samples from living patients should be to distinguish PD from AD patients and controls [24]. At present, formally assessed by an expert using standardized criteria (i.e., UK however, most potential biomarkers, have not achieved a level of Parkinson’s Disease Society Brain Bank), whereas neuropathological robustness high enough for routine clinical use and preliminary confirmation is mandatory for autopsy tissues. Patients’ medications results require further validation [25]. This leaves the door open and demographic data, such as age, gender, ethnicity, genetic back- for the design of innovative, sensitive and more targeted (i.e., post- ground, environmental and occupational specificities, should also translational modification detection) proteomic studies to identify be taken into account and groups of samples under study should be additional biomarkers of interest in CSF or other biological fluids. stratified accordingly. For example, as PD is a hetero­geneous dis- ease, it might be relevant to select patients with a similar phenotype “...proteomics has already allowed us to unravel a to identify markers that may vary according to clinical subtypes. number of pathways associated with Parkinson’s Furthermore, from a clinical point of view, it might be more rel- disease pathogenesis as well as potential evant to differentiate PD from other mimicking synucleinopathies Parkinson’s disease biomarkers...” than from AD or other phenotypically distinct neurodegenerative conditions. In addition to patient selection, sample quality is equally Direct analyses of autopsy samples from PD-affected struc- pivotal and many pre-analytical parameters have to be considered tures may provide a comprehensive, unbiased and unique view and monitored, including blood contamination, storage conditions of the degenerative mechanisms taking place in PD [17]. Thus and post-mortem delay, among others. far, proteomic studies have not only confirmed existing theories but also identified novel potential pathogenic molecules (for a “...both total DJ-1 and a-synuclein levels in human review see [17]), which, in turn, could also lead to the identifica- cerebrospinal fluid may represent useful tion of PD biomarkers. A firm advantage of post-mortem samples Parkinson’s disease diagnostic markers, when blood is that the diagnosis is definitely confirmed by neuro­pathological contamination and age factors are controlled.” examination. Moreover, regions of interest such as the SNpc can be precisely dissected out, cell populations as well as subcellu- In conclusion, we believe that neuroproteomics has an underesti- lar fractions isolated, and LBs purified for ana­lysis by sensitive mated potential to offer new, unbiased and highly sensitive strate- proteomic tools to unravel their proteomic specificities. Selecting gies to study the biological and molecular mechanisms underlying tissue samples might therefore be a way to address the difficult PD pathology and to identify diagnostic, prognostic or therapeutic question of pathology selectivity and cellular vulnerability. Some biomarkers. As PD is an exceedingly complex condition that can limitations that have been implicated against the use of such tis- only be partly modeled in the laboratory, we propose to tenaciously sues can now be overcome. Indeed, although the type, number, explore human samples from PD patients that, in the end, may quantity and quality of samples might be limited, access to represent the ideal material for basic and clinical research. them may be made easier through scientific collaborations with PD-experienced medical centers running brain donation programs Financial & competing interests disclosure and brain banks. Furthermore, recent studies have shown that Work performed by the authors in the field of this publication has been only a minority of proteins undergo massive degradation after generously supported by the Mémorial A de Rothschild foundation. The a prolonged post-­mortem delay of 72 h at room temperature [26]. authors have no other relevant affiliations or financial involvement with The problem of artifactual post-mortem changes can thus be cir- any organization or entity with a financial interest in or financial conflict cumvented within the range of a relatively long post-mortem delay. with the subject matter or materials discussed in the manuscript apart from Finally, the constantly evolving variety of proteomic techniques those disclosed. available, either gel-based (i.e., 2DE) or gel-free analyses involving No writing assistance was utilized in the production of this manuscript.

References 4 Brundin P, Li J-Y, Holton JL, Lindvall O, 6 Braak H, Del Tredici K, Rub U et al. Revesz T. Research in motion: the enigma Staging of brain pathology related to 1 Thomas B, Beal MF. Parkinson’s disease. of Parkinson’s disease pathology spread. sporadic Parkinson’s disease. Neurobiol. Hum. Mol. Genet. 16(R2), 183–194 (2007). Nature 9(10), 741–745 (2008). Aging 24(2), 197–211 (2003). 2 Poewe W. Non-motor symptoms in 5 Sulzer D. Multiple hit hypotheses for 7 Braak H, Ghebremedhin E, Rub U, Parkinson’s disease. Eur. J. Neurol. dopamine neuron loss in Parkinson’s Bratzke H, Del Tredici K. Stages in the 15(Suppl. 1), 14–20 (2008). disease. Trends Neurosci. 30(5), 244–250 development of Parkinson’s disease-related 3 Marsden CD. Parkinson’s disease. Lancet (2007). pathology. Cell Tissue Res. 318, 121–134 335(8695), 948–952 (1990). (2004). www.expert-reviews.com 293 Editorial Licker & Burkhard

8 Singleton A, Gwinn-Hardy K. Parkinson’s 14 Lees AJ, Hardy J, Revesz T. Parkinson’s 21 Tokuda T, Salem SA, Allsop D et al. disease and dementia with Lewy bodies: disease. Lancet 373(9680), 2055–2066 Decreased a-synuclein in cerebrospinal a difference in dose? Lancet 364(9440), (2009). fluid of aged individuals and subjects with 1105–1107 (2004). 15 Tanner CM. Advances in environmental Parkinson’s disease. Biochem. Biophys. Res. 9 Polymeropoulos M, Lavedan C, Leroy E epidemiology. Mov. Disord. 25(S1), 58–62 Commun. 349(1), 162–166 (2006). et al. Mutation in the a-synuclein gene (2010). 22 Mollenhauer B, Cullen V, Kahn I et al. identified in families with Parkinson’s 16 Consortium IPDG. Imputation of sequence Direct quantification of CSF a-synuclein disease. Science 276, 2045–2047 (1997). variants for identification of genetic risks by ELISA and first cross-sectional study in 10 Schiesling C, Kieper N, Seidel K, for Parkinson’s disease: a meta-analysis of patients with neurodegeneration. Exp. Kruger R. Review: familial Parkinson’s genome-wide association studies. Lancet Neurol. 213(2), 315–325 (2008). disease – genetics, clinical phenotype and 377(9766), 641–649 (2011). 23 Hong Z, Shi M, Chung KA et al. DJ-1 and neuropathology in relation to the common 17 Licker V, Kovari E, Hochstrasser DF, a-synuclein in human cerebrospinal fluid sporadic form of the disease. Neuropathol. Burkhard PR. Proteomics in human as biomarkers of Parkinson’s disease. Brain Appl. Neurobiol. 34(3), 255–271 (2008). Parkinson’s disease research. J. Proteomics 133(Pt 3), 713–726 (2010). 11 Clark LN, Wang Y, Karlins E et al. 73(1), 10–29 (2009). 24 Zhang J, Sokal I, Peskind ER et al. Frequency of LRRK2 mutations in 18 Beal MF. Parkinson’s disease: a model CSF multianalyte profile distinguishes early- and late-onset Parkinson disease. dilemma. Nature 466(7310), 8–10 (2010). Alzheimer and Parkinson diseases. Am. Neurology 67(10), 1786–1791 (2006). J. Clin. Pathol. 129(4), 526–529 (2008). 19 Potashkin JA, Blume SR, Runkle NK. 12 Ozelius LJ, Senthil G, Saunders-Pullman R Limitations of animal models of 25 Eller M, Williams DR. Biological fluid et al. LRRK2 G2019S as a cause of Parkinson’s disease. Parkinsons Dis. 2011, biomarkers in neurodegenerative Parkinson’s disease in Ashkenazi Jews. 658083 (2010). parkinsonism. Nat. Rev. Neurol. 5(10), N. Engl. J. Med. 354(4), 424–425 (2006). 561–570 (2009). 20 Hughes AJ, Daniel SE, Ben-Shlomo Y, 13 Wirdefeldt K, Gatz M, Schalling M, Lees AJ. The accuracy of diagnosis of 26 Crecelius A, Gotz A, Arzberger T et al. Pedersen NL. No evidence for heritability parkinsonian syndromes in a specialist Assessing quantitative post-mortem of Parkinson disease in Swedish twins. movement disorder service. Brain changes in the gray matter of the human Neurology 63(2), 305–311 (2004). 125(Pt 4), 861–870 (2002). frontal cortex proteome by 2-D DIGE. Proteomics 8(6), 1276–1291 (2008).

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Chapter IV

Proteomic profiling of the substantia nigra demonstrates CNDP2 overexpression in Parkinson's disease

The first experimental part of this thesis project consisted in the analysis of human substantia nigra (SN) in Parkinson’s disease and control patients by two-dimensional gel electrophoresis (2DE) technology, to gain new insights into PD pathogenesis. The proteomic analyses resulted in the representation of the most comprehensive 2DE reference map established so far, with more than 160 proteins and their isoforms identified. Interestingly, the comparative analysis demonstrated the deregulation of several proteins in PD, likely involved in neurodegeneration. In particular, we verified by orthogonal methods the overexpression of the poorly described dipeptidase cytosolic non specific dipeptidase 2 (CNDP2) and assessed its presence in dopaminergic neurons using immunological methods. Our findings support a role for the novel candidate CNDP2 in PD, through molecular mechanisms that still need to be determined.

I participated to the experimental part of the work presented in this chapter, and was responsible for all data analysis and interpretation. Moreover, I entirely wrote this original article published in 2012 in Journal of proteomics (IF=5.1).

JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667

Available online at www.sciencedirect.com

www.elsevier.com/locate/jprot

Proteomic profiling of the substantia nigra demonstrates ☆ CNDP2 overexpression in Parkinson's disease

Virginie Lickera, b, Mélanie Côtea, b, Johannes Alexander Lobrinusc, Neftali Rodrigoa, b, Enikö Kövarid, Denis F. Hochstrasserb, e, Natacha Turckb, Jean-Charles Sanchezb, Pierre R. Burkharda, b, f,⁎ aNeuroproteomics Group, University Medical Center, Faculty of Medicine, Geneva, Switzerland bBiomedical Proteomic Research Group, University Medical Center, Faculty of Medicine, Geneva, Switzerland cDepartment of Pathology, Geneva University Hospitals, Geneva, Switzerland dDepartment of Psychiatry, Geneva University Hospitals, Geneva, Switzerland eDepartment of Genetics and Laboratory Medicine, Geneva University Hospitals, Geneva, Switzerland fDepartment of Neurology, Geneva University Hospitals, Geneva, Switzerland

ARTICLE INFO ABSTRACT

Article history: Despite decades of intensive investigations, the precise sequence of molecular events and Received 25 November 2011 the specific proteins mediating the degenerative process underlying Parkinson's disease Accepted 25 February 2012 (PD) remain unraveled. Proteomic strategies may provide unbiased tools to identify novel Available online 6 March 2012 candidates and explore original mechanisms involved in PD. Substantia nigra pars compacta (SN) tissue, whose degeneration is the hallmark of PD, was dissected from neuro- Keywords: pathologically confirmed PD patients (n=3) and control subjects (n=3), before being submitted Parkinson's disease to a comparative 2-DE analysis. The present study revealed a subset of neuronal and/or glial Substantia nigra proteins that appears to be deregulated in PD and likely to contribute to neurodegeneration. Proteomics Observed alterations not only consolidate well accepted concepts surrounding PD pathogenesis 2-D gel electrophoresis such as oxidative stress and mitochondrial dysfunction but also point out to novel pathways. CNDP2 Among the latter, cytosolic non specific dipeptidase 2 (CNDP2), a relatively unknown protein not yet reported to be associated with PD pathogenesis, was shown to be increased in the SN of PD patients, as confirmed by Western blot. Immunohistochemical analyses demonstrated the presence of CNDP2 within the cytoplasm of SN dopaminergic neurons. Altogether, our findings support a key role of CNDP2 in PD neurodegeneration, by mechanisms that could involve oxidative stress, protein aggregation or inflammation. This article is part of a Special Issue entitled: Translational Proteomics. © 2012 Elsevier B.V. All rights reserved.

Abbreviations: CNDP2, cytosolic non specific dipeptidase 2; CSF, cerebrospinal fluid; DA, dopamine; LB, Lewy body; PD, Parkinson's disease; SN, substantia nigra pars compacta; UKPDSBB, United Kingdom Parkinson's disease society brain bank; WB, Western blot. ☆ This article is part of a Special Issue entitled: Translational Proteomics. ⁎ Corresponding author at: Department of Neurology, Geneva University Hospitals, 4, rue Gabrielle-Perret-Gentil, 1211 Geneva 14, Switzerland. Tel.: +41 22 372 83 09; fax: +41 22 372 83 32. E-mail address: [email protected] (P.R. Burkhard).

1874-3919/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jprot.2012.02.032 JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667 4657

has failed to decipher sporadic PD mechanisms. In this setting, 1. Introduction hypothesis-free strategies using novel high-throughput “omics” technologies may provide unbiased exploratory tools Parkinson's disease (PD) is a common and devastating neuro- to revisit the pivotal issues described above and identify degenerative disorder caused by the progressive loss of novel molecular pathways without relying upon pre-existing pigmented dopaminergic (DA) neurons in the substantia or a priori pathogenic hypotheses. Because proteins are the nigra pars compacta (SN) [1,2]. Lewy bodies (LB) – cytoplasmic major determinants of the diversity of phenotypes arising inclusions of aggregated proteins – and Lewy neurites are from a common set of genes and because sporadic PD can be observed in the surviving nigral neurons. Setting aside a essentially viewed as a disorder of protein handling, minority of PD cases who carry identified genetic mutations proteomics-based analysis might be one of the most appropri- or who have been exposed to neurotoxins such as 1-methyl- ate strategy to approach PD pathogenesis. 4-phenyl-1, 2, 3, 6-tetrahydroptyridine (MPTP) [3], the primary A limited number of proteomic studies investigating cause of PD neurodegeneration remains to be established. human post-mortem SN has been published so far in the field Sporadic PD is likely to result from the subtle interplay of PD research [10–13]. In the absence of fully satisfying PD between ageing, a predisposing genetic background and animal models, human autopsy tissues represent a unique environmental toxic factors, which converge to initiate a opportunity to highlight PD specific abnormalities. This cascade of still undetermined molecular events ultimately approach has been recently reappraised with the demonstration leading to cell death. Motor symptoms of PD (i.e. bradykinesia, that only a minority of proteins undergoes massive degradation rigidity or tremor) develop when about 60–70% of nigral after a prolonged post-mortem delay of 72 h at room temperature neurons are lost [4]. Therefore, in the absence of early [14]. The rationale for a whole tissue approach is provided by the biomarkers, PD diagnosis is still based on clinical manifestations fact that PD is an insidious disorder involving not only neurons that develop at an advanced neuropathological stage. So far, no but also their environment, within and beyond the SN, where treatment is effective to cure PD, and DA replacement therapies different subpopulations of glial cells (microglia, astrocytes, only provide a symptomatic relief at the expense of severe side oligodendrocytes) may contribute actively to DA neuronal effects. The limited understanding of PD pathogenesis and the death [15]. lack of reliable biomarkers constitute major hurdles for the In this study, SN was selectively dissected from PD development of strategies able to slow or halt disease patients' and controls' autopsy tissues and extracted proteins progression. were submitted to two-dimensional gel electrophoresis In the last decades, genetic studies have provided some aid (2-DE). We delineated a subset of deregulated neuronal and in understanding the mechanisms underlying the condition. glial proteins in PD which may participate to the pathological For example, genes harboring mutations causally related to processes related to PD. Observed alterations consolidate rare monogenic forms of PD have suggested that deregulations some popular theories such as oxidative stress and mitochon- in alpha-synuclein (α-SYN,SNCA) metabolism, mitochondrial drial dysfunction but also point out to yet unidentified path- functions (PINK1, DJ-1, LRRK2), protein degradation (Parkin, ways. Novel potentially pathogenesis-relevant candidates were UCHL-1) or antioxidant (DJ-1) systems (reviewed in [5])maybe identified, such as the cytosolic non specific dipeptidase 2 central in PD pathogenesis. The involvement of those proteins (CNDP2) which appears to be overexpressed in PD nigral or pathways in sporadic PD, which represents about 90% of all neurons. PD cases, was further substantiated by various observations. Recently, genome wide association studies have provided evidence that common variants in several genes – including 2. Materials and methods some known from inherited PD subtypes (i.e. SNCA or LRRK2) and others (i.e. MAPT) – were conferring an increased suscepti- 2.1. Human samples bility to develop sporadic PD [5]. Examination of sporadic PD patients' SN showed a reduced activity of the ubiquitin protea- 2.1.1. Brain tissues some system (UPS) and mitochondrial complex I as well as Mesencephalon specimen from six PD patients and four higher concentrations of oxidized proteins [6,7]. Importantly, control subjects were collected at autopsy in the Division of LBs in sporadic PD brains are strikingly positive for α-SYN and Clinical Pathology of the Geneva University Hospitals under ubiquitin [8,9]. Whether LB themselves cause neurodegenera- an ethically approved protocol and either frozen at −80 °C or tion is still unclear, but abnormal accumulation of unwanted fixed in 15% formaldehyde for 4 weeks at 4 °C before being proteins and failure of the UPS to degrade them, successively paraffin-embedded (Table 1). An informed consent form was leading to protein oligomerization and aggregation, appears signed by close relatives to proceed with the protocol central to cell demise. Furthermore, growing evidence has sug- research. PD final diagnosis was confirmed post-mortem by gested that protein misfolding, mitochondrial dysfunction and neuropathological examination assessing the presence of two altered autophagy of mitochondria, oxidative stress, energy pathological hallmarks of the condition, i.e. severe neuronal production imbalance, excitotoxicity, inflammation, defects in loss in the SN and presence of α-SYN immunoreactive inclu- neurotrophic factors, or apoptosis are all contributing factors. sions (LB). Controls were cases with no previous history of any However, neither unifying nor completely satisfying hypothe- neurological or psychiatric disorders and no nigral abnormalities. ses on PD pathogenesis have been established yet. For all experiments, patients from both groups were matched as Mainly driven by genetic discoveries, candidate-based closely as possible with respect to age, gender and post-mortem research focusing on selected pathophysiological pathways interval (<34 h). 4658 JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667

Table 1 – Clinicopathological data of human post-mortem samples. Abbreviations: DD=disease duration, PMI=post-mortem interval, SD=standard deviation.* indicates paraffin-embedded mesencephalons, whereas others are frozen tissues. Experiment ID Gel n° Diagnosis Gender Age (y) DD (y) PMI (h)

C1 107 Ctrl M 85 12 2-DE/WB C2 109 Ctrl M 88 18 C3 110 Ctrl M 77 30 Mean (±SD) 3 M 83.3 (±5.7) 20 (±9.2) P1 103 PD M 82 5 28 2-DE P2 104 PD M 84 7 24 P3 105 PD M 79 19 7 Mean (±SD) 3 M 81.7 (±2.5) 10.3 (±7.6) 19.7 (±11.2) P2 / PD M 84 7 24 WB P4 / PD M 77 2 25 P5 / PD F 85 2 24 Mean (±SD) 2 M/1 F 82.0 (±4.4) 3.7 (±2.9) 24.3 (±0.6) IHC C4 * / Ctrl F 92 34 P6 * / PD M 73 16 25

For SN dissection, frozen tissue was equilibrated 30 min at the United Kingdom PD Society Brain Bank (UKPDSBB) criteria −20 °C before cryosection of 18 μm-thick slices on glass slides [16]. A neuropathological examination was later performed in 3 and immediate processing. Slides were kept on ice and dried at out of these 7 cases, confirming PD diagnosis. Age/sex-matched room temperature (20 °C on average) 1 min before dissection. control subjects consisted of healthy individuals with no sign or The SN was identified macroscopically by its dark history of any neurologic disorders. All clinical details are neuromelanin-enriched crescent-shape pigmentation and summarized in Table 2. Each patient gave informed consent microscopically by the presence of DA neurons observed prior to enrollment and all protocols were approved by the after staining. The area of interest could be local institutional ethical committee board. All CSF samples easily identified even in PD cases that exhibited an apparent were cleared for 10 min at 3000 g by centrifugation at 4 °C, before loss of pigmentation, and selectively dissected from slides being stored at −80 °C. with a scalpel. Scraped tissue pieces were transferred into cold Eppendorf tubes and weighted until 1 mg of nigral tissue 2.2. 2-DE analysis was obtained. Samples were kept at −80 °C before further processing. 2.2.1. Sample preparation and 2-DE gels Three PD and three control cases (Table 1) were analyzed by 2.1.2. Cerebrospinal fluid 2-DE, with all gels being run in the same experiment. For Cerebrospinal fluid (CSF) samples were collected by routine large 2-DE analytical gels, 1 mg of each sample was solubilized lumbar puncture in the Department of Neurology of the Geneva with 400 μL of rehydration buffer (urea 7 M, Thiourea 2 M, Tris University Hospitals from patients with typical, levodopa- 30 mM, DTE 65 mM, CHAPS 4%, anti-proteases, 2% ampholines responsive PD (n=7) and control (n=7) cases. PD diagnosis was 3–10 and traces of bromophenol blue), vortexed for 1 h at room assessed by a movement disorder specialist strictly applying temperature (20 °C on average) and loaded onto a commercial

Table 2 – Clinical data of human CSF samples. Abbreviations: DD=disease duration at lumbar puncture, SD=standard deviation, PM=postmortem. ID Diagnosis Gender Age (y) DD (y) PM diagnosis confirmation

C5 Ctrl F 69 C6 Ctrl M 77 C7 Ctrl F 76 C8 Ctrl M 71 C9 Ctrl M 67 C10 Ctrl M 76 C11 Ctrl F 72 Mean (±SD) 4M/3F 72.6 (±3.9)

P7 PD M 68 12 / P8 PD M 68 15 yes P9 PD F 44 10 / P10 PD M 69 5 / P11 PD M 70 2 yes P12 PD M 71 10 / P13 PD F 76 10 yes Mean (±SD) 5M/2F 66.6 (±10.3) 9.1 (±4.3) JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667 4659

18-cm pH 3–10 non-linear IPG strip (GE healthcare) by over- system (Waters Corporation, Milford, MA). Peptides were night (18 h) in-gel rehydration. The electrophoretic separation separated for 30 min on a home-made analytical column – was performed as previously described [17 19]. Vertical gradi- using a gradient of 99.9% H2O, 0.1% FA (solvent A) and 99.9% ent gels (8–16% T, 2.6% C) were used for the second dimension. ACN, 0.1% FA (solvent B). Mass spectra were acquired in the pos- Proteins were fixed and detected using a sensitive ammoniac itive mode within a window of 400–2000 m/z. Four precursors silver stain [20]. Preparative 2-DE gels were obtained by loading were selected for collision-induced dissociation (CID) in the a higher amount (generally 2 mg) of control or PD case tissues LTQ (isolation width of 2 m/z). The normalized collision to generate spots with enough material for MS identification energy was set to 35%. and by using a modified MS-compatible staining procedure [21]. 2.2.5. Protein identification Peak lists were generated using either the 4000 Series Explorer 2.2.2. 2-DE image analysis software from Applied Biosystems (MALDI TOF/TOF), or the Scanned gel images were analyzed with ImageMaster™ 2D embedded software (extract MSN.exe) from Thermo Electron Platinum software v5.01 (GE healthcare) for automatic spot (LTQ-OT). Peak lists were searched individually against detection and quantification. For each individual spot, Uniprot-Swissprot (57.11 of November 24 2009) database percentage volumes (%vol) were computed to correct for restricted to Homo Sapiens taxonomy using Mascot (version differences in sample loading or staining. Analysis was 2.2, Matrix Science, London, UK) when acquired with a performed by matching each individual gel to the reference MALDI-TOF/TOF instrument or with Phenyx (version 2.6, gel, which was in turn a PD gel (sample P2, gel 104) and a GeneBio, Geneva, Switzerland) when acquired with an LTQ- control gel (sample C2, gel 109) to ensure the detection of OT. Using Mascot, monoisotopic mass, with precursor mass group-specific spots. PD/C ratios were calculated for each error of 400 ppm and fragment mass tolerance of 0.4 Da were spot on the basis of corresponding mean %vol, as well as selected. Peptides with ion scores above the identity threshold intra-group coefficient of variation (CVC, CVPD). As no robust were selected, assuming 5% probability of false match. statistical test applies to such a small number of samples, With Phenyx, the minimum peptide length was five amino we used stringent criteria (CV, ratio and/or t-test) to select acids and precursor error tolerance was 10 ppm. False positive differentially expressed spots. First, we made the assumption ratios were estimated using a reverse decoy database [23]. All that for a spot to be different between groups, biological datasets where searched once in the forward and once in the variability assessed by intra-group CVs, should be smaller reverse database. Peptide z-scores were then set to maintain than the variability between the two groups given by: if ratio a false positive peptide ratio below 1%. For both Mascot and − >1: CVC +CVPD <(ratio 1)*100 or if ratio <1: CVC +CVPD <((1/ Phenyx search, trypsin was selected as the proteolytic ratio)−1)*100). In addition, spots with a ratio of mean above enzyme, one missed cleavage was allowed, carbamidomethyla- the 1.4 threshold (or <0.72) with or without a significant statis- tion of cysteines was set as a fixed modification whereas oxidized tical unpaired Student t-test (p<0.05) were retained. Finally, methionine variable. Proteins with at least two different peptide retained spots were visually checked and only well focalized sequences were kept. and defined spots were selected for MS identification. 2.3. Western blot (WB) 2.2.3. In gel digestion of 2-DE spots Protein spots were excised manually from the preparative gels The expression level of cytosolic non specific dipeptidase 2 and destained before being subjected to in-gel tryptic digestion (CNDP2) was assessed by WB in the SN extracts and CSF. For and peptide extraction using previously described protocols SN samples, 1 mg of tissue was solubilised in sample buffer [22]. Identification was performed by tandem mass spectrometry (0.1 M TEAB pH 8.0, 0.1% CHAPS, 0.05% SDS and a protease (MS/MS) either using a MALDI-TOF/TOF™ for235spotsoralinear inhibitor cocktail from Roche) and vortexed for 1 h at room trap quadrupole orbitrap (LTQ-OT) for the 23 spots differentially temperature. Protein concentrations were determined by expressed spots. Bradford Assay (Bio-Rad). Either 10 μg of nigral proteins from each patient (C1–C3, P2, P4 and P5) or 10 μg of total CSF protein 2.2.4. MS/MS analysis (C5–C11, P7–P13) were separated by 12.5% T SDS-PAGE and For MALDI-TOF/TOF MS analysis, samples were desalted, transferred onto nitrocellulose membranes. Immunoreactive diluted in 5% ACN, 0.1% TFA and spotted in duplicates onto bands were detected by ECL Western blotting system (GE an Opti-TOF™ plate. Identical volumes of matrix (5 mg/mL α- Healthcare). Mouse monoclonal antibodies against CNDP2 cyano-4-hydroxycinnamic acid in 50%ACN, 0.1%TFA, 10 mM from Abnova were used at a final concentration of 2 μg/ml

NH4H2PO4) were added to the previously loaded digest. Mass on SN tissue. Mouse monoclonal antibodies from R&D were spectra were acquired with a 4800 MALDI-TOF/TOF analyzer used at a final concentration of 1 μg/ml. Anti-mouse horseradish (Applied Biosystems, Foster City, CA) using the positive ioniza- peroxidase-conjugated secondary antibodies (Dako) were dilut- tion mode and a scan range of 800–4000 m/z. The 15 most in- ed at 1:1000. Images were analyzed by TotalLabQuant ™ (GE tense peaks with signal-to-noise ratio over 10 were subjected Healthcare) to determine band volumes. to MS/MS analysis. Argon was used as collision gas. For electrospray ionization (ESI) LTQ-OT MS analysis, 2.4. Immunohistochemistry samples were dissolved in 5% ACN, 0.1% formic acid (FA) and analyzed on a LTQ Orbitrap XL mass spectrometer (Thermo Formaldehyde-fixed (15%), paraffin embedded human autop- Electron, San Jose, CA) equipped with a NanoAcquity HPLC sy tissues from the SN of patients C4 and P6 were sectioned 4660 JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667 at 12 μm on gelatin-coated slides. Mounted sections were with Cresyl Violet. Negative controls were performed by hy- deparaffinized in xylene and dehydrated in ethanol (60°). Neu- bridization with the secondary antibody only. romelanin pigmentation was bleached with 0.25% potassium permanganate in PBS for 10 min and 1% potassium bisulfite/ 1% oxalic acid in PBS for a few minutes. Sections were incu- 3. Results bated overnight at 4 °C with mouse monoclonal primary anti- body against CNDP2 (Abnova), diluted 1:200 in 0.3% Triton 3.1. 2-DE map of SN tissue X-100/ 1% BSA in PBS. Sections were then incubated for 1 h at room temperature with anti-mouse horseradish peroxidase- The representative 2-DE protein map of human SN is conjugated secondary antibody (Dako), diluted 1:100 in 0.3% shown in Fig. 1. MS analysis allowed the identification of a Triton X-100/ 1% BSA in PBS. 3,3′-diaminobenzidine (DAB) was total of 257 spots, corresponding to 163 proteins, listed in the used as chromogen (Sigma). Sections were counterstained Supporting Files 1 and 2. Sometimes, more than one protein

Fig. 1 – 2-DE map of the human SN proteome. Representative gel image (gel 104, patient P2) of protein pattern in the SN. One mg SN tissue was loaded on an IPG strip (pH 3–10, NL, 18 cm). The second dimension was performed on a vertical gradient slab gel (8–16% T). Gels were visualized using silver staining. Gene names of identified proteins are indicated on the gel, with corresponding spot numbers listed in supporting file 2. Protein names and MS data are given in supporting file 1 and 2, respectively. JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667 4661 was identified in a spot, especially when using the more of interest (i.e. mitochondrial inner membrane protein) we de- sensitive LTQ-OT instrument. cided not to comment further on them prior any supplementary verification indicating which protein was responsible for the ex- 3.2. Detection of differentially expressed spots in the SN of pression level change. PD patients by 2-DE analysis 3.3. WB verification of CNDP2 overexpression in the SN of Protein extracts from the SN of PD (n=3) and control (n=3) PD patients patients were separated in parallel by 2-DE. Macroscopically, no major differences in spot patterns were observed between Among the differentially expressed proteins identified in both groups. A linear regression analysis plotting inter-gels 2-DE, we chose to verify cytosolic non specific dipeptidase %vol was performed as in Carrette et al. [24] to assess inter- (CNDP2). The reasons for further analysis of CNDP2 are as gel variability. Table 3 summarizes the resulting data (i.e. follows: (1) CNDP2 exhibited one of the highest calculated best fit line equation parameters, R2). R2 coefficient values be- ratios of all differentially expressed proteins; (2) CNDP2 is tween 0.82 and 0.93 signify a high correlation of spots %vol on overexpressed in PD patients, rather than underexpressed matched gels. Best fit lines were close from identity (1.0 x+0), which could derive from neuronal cell loss; (3) CNDP2 had indicating that most of the protein spots were equally one of the highest %vol value of all differentially expressed expressed in different conditions (inter- or intra-class). Intra- spots; (4) CNDP2 is a previously unreported pathogenic candi- class linear regression analyses demonstrated a high level of date with no known link with PD so far. Using a 2-DE ap- homogeneity, with low global scale variability in terms of proach, two CNDP2 spots – (a) and (b) – were identified and %vol, of a maximum of 6% in the PD class and 23% in the Con- only spot (b) (no 3 in Table 4a) was shown to be overexpressed trol class. Therefore, the cutoff threshold to consider a spot in the SN of PD patients at a PD/C ratio of 2.5, as represented in differentially expressed between classes was determined as Fig. 3A. WB detection of CNDP2 with a monoclonal commer- 1+(max variability PD+control)=1+0.29=1.3. To be more cially available antibody (Abnova) clearly confirmed a stringent, only ratios over 1.4 or below 0.72 were considered. significant increase of the protein level in PD patients. Similar Inter-class linear regression analyses also pointed toward to 2-DE analysis, a PD/C ratio of 3.1 was calculated using actin a very low variability level, indicating that differences in a few normalized band volumes, as represented in Fig. 3B. spots only, are expected to be characteristic of each state. Compared to CNDP2 spot (a), differential spot (b) was We compared proteomic patterns between gel classes slightly shifted in the lower molecular weight and more using the spot % volumes calculated by the software. Thirty- basic pH (Fig. 3A, lower panel). Mass shift might be too small two spots met our criteria to be differentially expressed in to be resolved on 12.5% SDS-PAGE gel and a unique band PD compared to control groups. These spots are listed in combining both spots was observed on WB membrane. It is Table 4 and mapped on Fig. 2 where spots increased in the also possible that for some reasons, CNDP2 antigen of the PD group are highlighted in red and spots decreased spots in stable CNDP2 spot (a) form is hidden and not recognized by blue. Details on selected spots including individual spot %vol the antibody. CNDP2 spot (b) staining pattern in control across gels, intra-class CVs, ratios and p-values are given in patients is far weaker than spot (a). All together, those obser- Supporting File 3. Fourteen spots were increased in the PD vations indicate a shorter, potentially pathogenic CNDP2 group (Table 4a) whereas 18 were decreased (Table 4b). Of form in PD patients that could result from protein truncation them, 23 were successfully identified by LTQ-OT or MALDI or PTM cleavage, different alternative splicing products or TOF-TOF instruments, corresponding to distinct identifications. nonsense mutation, as examples. In 6 spots (12, 13, 18, 20, 25, 30) we identified two to three pro- teins that could be responsible individually or in combination 3.4. Immunohistochemical analysis of CNDP2 in SN for the ratio change. Thus, although some of them seem to be tissues

To further validate our 2-DE and WB results, we performed an Table 3 – Results of the linear expression study of gels. Value of R2 indicates the quality of the fit. Inter-gel global immunohistochemical (IHC) analysis on paraffin-embedded scale variability in %vol is calculated as (a−1)×100. PD PD nigral tissues of a PD and a control patient (Table 1), using a class, C Control class. Bold lines indicate gels from the CNDP2 monoclonal antibody (Abnova) (Fig. 4). Because dense same class. neuromelanin aggregates hinder the intraneuronal immuno- RefGel vs ab(a−1)×100 R2 # matched detection of CNDP2, samples were bleached by a potassium Gel 2 spots permanganate treatment. No staining was observed in the negative controls (data not shown). 104PD vs 107C 0.92 0.0047 −8% 0.882 1268 When comparing mesencephalic staining patterns at low 104PD vs 109C 0.98 0.00035 −2% 0.929 1018 104PD vs 110C 1.25 −0.017 +25% 0.821 1208 magnification (data not shown), PD patient exhibited a more 104PD vs 103PD 1.06 −0.0023 +6% 0.904 1281 intense staining than controls, principally in the SN. In both 104PD vs 105PD 0.98 −0.00062 −2% 0.925 1268 cases, a light staining outside the SN was observed, indicating 109C vs 103PD 1 0.00097 0% 0.895 1071 that CNDP2 expression might not be restricted to SN. Interest- C PD − 109 vs 105 0.97 0.00078 3% 0.909 1122 ingly, CNDP2 was definitely detected in DA neuronal popula- 109C vs 104PD 1 0.00039 0% 0.929 1021 109C vs 107C 0.88 0.0056 −12% 0.897 1079 tions, yet probably expressed in specific glial cell populations 109C vs 110C 1.23 −0.014 +23% 0.854 1055 as well (data not shown). Thus, CNDP2 might not be a neuron-specific protein. At higher magnification (Fig. 4), the 4662 JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667

Table 4 – a. 2-DE spots overexpressed in PD patients b. 2-DE spots underexpressed in PD patients. Panel a

Mean %vol±SD

spot PD Control PD/C AC Protein name MW pI

1 0.056±0.010 0.001±0.000 57.61 No ID 2 0.030±0.004 0.006±0.009 4.7 P62873 Guanine nucleotide-binding protein G(I)/G(S)/G(T)β-1 37353 5.6 3 0.152±0.042 0.061±0.042 2.47 Q96KP4 Cytosolic non-specific dipeptidase 52747 5.7 4 0.007±0.001 0.004±0.001 1.72 Q9NRX4 14 kDa phosphohistidine phosphatase 13833 5.9 5 0.041±0.008 0.024±0.003 1.7 Q9UBQ0 Vacuolar protein sorting-associated protein 29 20506 6.5 6 0.007±0.001 0.005±0.001 1.66 No ID 7 0.037±0.001 0.023±0.003 1.63 P61088 Ubiquitin-conjugating enzyme E2 N 17007 7 8 0.014±0.003 0.009±0.001 1.62 P04179 Superoxide dismutase [Mn], mitochondrial 22204 7.3 9 0.497±0.061 0.314±0.035 1.58 P02792 Ferritin light chain 19888 5.6 10 0.02±0.003 0.012±0.001 1.57 P12277 Creatine kinase B-type (fragment) 42513 5.4 11 0.060±0.013 0.039±0.009 1.51 Q9GZN8 UPF0687 protein C20orf27 19160 6.5 12 0.017±0.002 0.012±0.001 1.41 Q16718 NADH dehydrogenase 1 α subunit 5 13328 6.2 P06702 Protein S100-A9 13111 5.9 13 0.220±0.006 0.157±0.036 1.41 Q16555 Dihydropyrimidinase-related protein 2 62255 6 14 0.147±0.024 0.105±0.008 1.39 Q16352 Alpha-internexin 55391 5.4 P10809 60 kDa heat shock protein, mitochondrial 57963 5.3

Panel b

15 0.001±0.000 0.009±0.004 0.11 No ID 16 0.030±0.050 0.145±0.039 0.2 No ID 17 0.002±0.002 0.007±0.001 0.35 No ID 18 0.068±0.021 0.178±0.058 0.38 P08133 Annexin A6 75742 5.5 P11142 Heat shock cognate 71 kDa protein 70767 5.4 P54652 Heat shock-related 70 kDa protein 2 70021 5.6 19 0.013±0.011 0.035±0.004 0.39 No ID 20 0.008±0.004 0.020±0.006 0.39 P01024 Complement C3c alpha' chain fragment 2 71317 7.1 P15924 Desmoplakin (DP) (fragment) 331774 6.5 21 0.078±0.026 0.173±0.030 0.45 O75947 ATP synthase subunit d, mitochondrial 18360 5.4 22 0.019±0.011 0.040±0.010 0.46 P04181 Ornithine aminotransferase, mitochondrial 45853 6.1 23 0.105±0.045 0.216±0.066 0.49 No ID 24 0.037±0.014 0.069±0.006 0.54 P09972 Fructose-bisphosphate aldolase C 39325 6.8 25 0.044±0.005 0.08±0.021 0.55 P14618 Pyruvate kinase isozymes M1/M2 58062 8 P27338 Amine oxidase [flavin-containing] B 58632 7.6 P30038 Delta-1-pyrroline-5-carboxylate dehydrogenase, mit 59034 7.3 26 0.081±0.008 0.146±0.023 0.56 P22695 Cytochrome b-c1 complex subunit 2, mit 46784 8.3 27 0.008±0.002 0.014±0.003 0.58 Q15700 Disks large homolog 2 (fragment) 83405 5.8 28 0.021±0.005 0.035±0.006 0.62 No ID 29 0.040±0.003 0.06±0.004 0.66 No ID 30 0.018±0.003 0.027±0.005 0.67 Q16891 Mitochondrial inner membrane protein 82625 6.2 P06396 Gelsolin (ADF) 80641 5.6 31 0.022±0.001 0.033±0.005 0.68 P61764 Syntaxin-binding protein 1 67569 6.6 32 0.072±0.004 0.099±0.012 0.72 Q99747 Gamma-soluble NSF attachment protein 34746 5.4

major difference between pathological and healthy states and healthy control subjects (n=7) (Table 2) by WB analysis, seems to reside in the staining level of DA neurons, more using a CNDP2 monoclonal antibody (R&D). CSF was loaded intense in the PD case (Fig. 4-b, 4-d, arrows). Neuronal labeling on 12.5% polyacrylamide gels in equal total protein amounts appears to be cytosolic, with a staining distribution inside the based on Bradford determination (10 μg). In all cases, a double whole cell, as shown in other cell types and species (www. band was detected around 54 kDa (Fig. 5). It was not possible to proteinatlas.org, [25]). Interestingly, we noticed a granular quantify the two bands independently, but visually, no major staining pattern that could be indicative of CNDP2-containing “band specific” difference seemed to emerge between groups vesicles or CNDP2 multiple aggregates (data not shown). on visual assessment. Quantified band volumes were found not to be significantly differentially expressed between groups 3.5. CNDP2 detection and measurement in the CSF of PD (p>0.05, Mann–Whitney U test), as shown in Fig. 5. WB analysis patients was also performed using equal volumes (14ul) of CSF, which gave similar results (data not shown). Of note, the monoclonal We further examined whether CNDP2 could be detected in the anti-CNDP2 antibody specificity was confirmed by WB analysis CSF of living patient and ultimately serve as a novel biomarker using recombinant human CNDP1 and CNDP2 proteins (data of PD. For that purpose, we tested the CSF of PD patients (n=7) not shown). JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667 4663

Fig. 2 – Representative PD reference gel showing all differentially expressed spots. One mg nigral tissue from PD (n=3) and C (n=3) patients was subjected to 2-DE as previously described (Fig. 1). Thirty-two spots were found to be differential in the two groups and are reported on the gel. Fourteen spots were increased (red) in PD whereas 18 were decreased (blue) with ratios over 1.4 or below 0.72 respectively. Of them, 23 were identified, as reported in Tables 4a and 4b.

Fig. 3 – CNDP2 overexpression in the SN of PD patients. A: Comparative 2-DE analysis. Upper panel shows quantification data of the two CNDP2 spots (a) and (b) identified in the 2-DE study, calculated using spot %vol in the six gels and given in % of controls. Bars represent mean with SD. Spot (a) did not exhibit any expression difference between groups, whereas spot (b) or spot no. 3 in Table 4a, is significantly increased in PD patients SN with a PD/C ratio of 2.5 (p<0.05, unpaired t test). Representative enlargement of control (gel 109, C2) and PD (gel 104, P2) gels are shown in the lower panel. White arrows point to CNDP2 spot (a) whereas black and red arrows point to differential CNDP2 spot (b). B: Western blot analysis. CNDP2 band volumes were normalized using β-actin and are given in % of controls. CNDP2 level is significantly increased in PD patients' SN with a PD/C ratio of 3.1 (p<0.05, unpaired t test). Bars represent mean with SD. 4664 JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667

Fig. 4 – Immunohistochemical detection of CNDP2 in nigral neurons. Representative anti-CNDP2 immunohistochemistry of SN, showing the expression of the protein in the SN of a PD patient (c–d) and a control subject (a–b). Staining pattern in brown (DAB) seems to be predominant in DA neurons (i.e. arrows in b, d) recognizable by their size. Staining pattern inside cells points to a main cytosolic expression. a and c: 5× magnification, b and d: 20× magnification.

4. Discussion

4.1. Deregulated set of proteins identified in the SN of PD patients

So far, only a small number of proteomic studies exploring the SN of PD patients has been published, four to the best of our knowledge [10–13]. This probably reflects the difficulty to obtain human tissues, although this type of sample represents, in our view, a source of choice to detect PD specific abnormali- ties [26]. Of them, only two used a 2-DE approach [12,13].In the first study by Basso et al., 44 nigral proteins were identified from which 9 were found differentially expressed, whereas in that by Werner et al., 37 proteins were identified from which 16 – on the 221 differentially expressed spots detected – were found altered. In this report, we further expanded our knowl- edge of the SN proteome 2-DE map, with a total of 163 proteins and some of their isoforms identified, mainly corresponding to new protein identifications (112 on 163). Two-DE comparative Fig. 5 – CNDP2 detection in the CSF of PD vs control individuals. analysis of selectively dissected nigral tissue samples allowed WB analysis showing CNDP2 expression in the CSF of control the detection of 32 differentially expressed spots in PD patients subjects (n=7) and PD (n=7) patients. An equal total amount of of which 23 were identified by MS/MS. A majority of the differ- proteins (10 μg) of each CSF sample (n=14) were separated on entially expressed spots identified (15 out of 23) were novel a 12.5% polyacrylamide gel. Mean of quantified band volumes candidate proteins as they have not been identified by the are represented with their respective error (SD). No statistically other groups. significant differences (Ns) were observed between the Control Although a high proportion of non-differential proteins and PD groups (p value>0.05, Mann–Whitney U test). WB were concordant across the proteomic studies cited above, analysis was also performed using equal volumes (14 μl) of only few proteins were found similarly differential. For example, CSF, which similarly yielded statistically not significant results ferritin was also found increased in Werner et al., but the heavy (data not shown). chain and not the light chain as in our report. ATP synthase JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667 4665 subunit D was found increased in Basso et al. whereas we found functions and their potential association with PD pathogene- it decreased, which can be explained by the fact that different sis is given in Supporting File 5. We found a differential protein isoforms, inversely expressed, can be involved in PD expression in multiple proteins previously linked to mito- pathogenesis by different mechanisms. PD is known to be a chondria, oxidative stress and energy metabolism impair- heterogeneous disease and for example, duplication or triplica- ments, three interconnected processes that have long been tion of α-SYN as well as point mutations which do not increase known to be associated with neuronal demise in PD patho- α-SYN load, result in the same PD phenotype [27,28]. genesis (for review see [30]). Components of the mitochondrial In our view, each 2-DE experiment might show a unique respiratory chain complexes III (cytochrome b–c1 subunit 2) picture of the nigral proteome, revealing previously unde- and V (ATP synthase subunit D) were found markedly tected or unresolved spots depending on various potentially decreased in PD brains, confirming a reduced mitochondrial biasing modifiers. Our study differs from others in many activity that may be pathogenic by itself or related to mito- ways, including subject determination, sample quality and chondrial complex I defects observed in PD brains [7,31]. preparation as well as analysis and identification methods. Superoxide dismutase [Mn] and Ferritin L-chain, both impli- For example, we chose to dissect SN slice by slice (18 μm cated in cell protection against free radical damages, were each) to be highly selective, whereas others have preferred found increased in PD brains. This is consistent with a cellular to homogenize a whole specimen at the risk of including response to increased oxidative stress as observed in PD disease-unrelated adjacent tissues. We set up an experimental brains [32]. The glycolytic enzyme fructose-biphosphate aldol- workflow ensuring a good reproducibility and high sensitivity ase C was found decreased in PD, pointing to a reduction of in spot pattern detection, for example by performing a single energy metabolism and ATP depletion. Creatine kinase B, 2-DE run with all the samples and using silver staining. The involved in sub-cellular energy transport and distribution use of more sensitive MS instruments allowed reliable identifi- between ATP production sites was found to be increased, cations by MS/MS and sometimes multiple protein identifica- which may indicate compensatory mechanisms at work to tions in a spot. The careful selection of differentially counteract local energy deficiency. DA neurons may be more expressed spots by stringent criteria was somehow validated vulnerable than any other cells to energy metabolism impair- with the confirmation of CNDP2 overexpression by an orthog- ment, but also to any form of insult, as they require higher onal method (WB). Supplemental file 4 provides an extensive energy production to achieve basic functions, particularly comparison of the many methodological parameters that axonal transmission [29]. vary across the different 2-DE studies and could account for Our data also highlighted changes in proteins previously the lack of concording results. not associated with PD such as the guanine nucleotide binding Using a whole tissue approach, we analyzed the global protein G(I)/G(S)/G(T) subunit beta, CNDP2, 14 kDa phospho- protein expression from all different cell types present in the histidine phosphatase or vacuolar protein sorting-associated SN. Thus, except for characterized cell-type specific proteins, protein 29 (VPS29) all increased in the PD SN. Since G- observed protein changes could be related to both neuronal proteins are regulators of multiple signaling cascades, guanine and glial cells. A few observations indicate that we might nucleotide binding protein G(I)/G(S)/G(T) subunit beta may have observed at least some neuronal changes. First, the play an important role in mediating neuronal functions majority of protein ratios fell between 1.5 and 2, which through several intracellular pathways. Its overexpression account for relatively small changes. This is consistent with may lead to altered signaling pathways in DA neurons and changes detectable from DA neurons. Indeed, DA neurons subsequent neurodegeneration. VPS29 is part of the retromer representing approximately 4 to 10% of SN cells in PD and cargo recognition complex, which is central for membrane- control patients, their signal is expected to be diluted at least associated proteins recycling and endosomal-lysosomal traf- 10 times and hardly detectable. Second, no difference in ficking. Interestingly, a mutation in VPS35, another retromer glial-specific proteins was detected but at least two strictly component tightly linked to VPS29, was recently discovered neuronal proteins involved in synaptic functions, syntaxin in a kindred exhibiting an inherited form of PD [33]. binding protein 1 and disk large homolog 2, were found to be decreased in PD SN, probably as a result of neuronal loss and 4.2. CNDP2 as a potential novel candidate in PD pathogenesis unrelated to abnormalities in any pathogenic pathways. In contrast, protein overexpressions in the SN of PD patients In this study, using a 2-DE approach, we observed an over- could result from their neuronal accumulation in a potentially expression of CNDP2 in the SN of PD patients, a finding that toxic or inefficient form and eventually in LBs. Of note, classical was validated by WB and supported by IHC observations. PD-associated neuronal α-SYN increase was not observed here, To the best of our knowledge, this report is the first propos- unless α-SYN is one of the unidentified spots reported in ing an involvement of CNDP2 in PD pathogenesis, as no previ- Table 4. α-SYN could also be undetected in 2-DE due to its low ous studies have ever described its association with any other expression level which might not fit into 2-DE dynamic range, neurodegenerative disease. So far, very few information is and its biochemical properties or aggregated state that might available about this protein. CNDP2 was shown to be ubiqui- have precluded its correct solubilization, 2-DE migration and tously expressed throughout human tissues whereas its ho- focalization. mologue CNDP1 is secreted and preferentially expressed in Interesting links can be established between some poten- the brain [34]. Both proteins belong to the M20 metallopro- tially relevant candidate proteins identified in this study and tease family and have been shown to carry a carnosine (β- previously known mechanisms associated to PD neurodegen- alanyl-histidine) peptidase activity [34,35]. As recently dem- eration [29]. A table summarizing the differential protein onstrated, CNDP2 cleaves other dipeptides with high affinity, 4666 JOURNAL OF PROTEOMICS 75 (2012) 4656– 4667 such as Cys-Gly of the γ-glutamyl cycle, participating in gluta- CNDP2 expression in an oxidative stress model of PD human thione (GSH) biosynthesis [36]. Both carnosine and glutathi- SH-SY5Y cell lines (i.e. rotenone). Secretion tests could be one are pivotal free radical scavengers, which could exert a performed to determine if CNDP2 is expressed in this cell-line neuroprotective activity through their ability to counteract and subjected to variations depending on conditions. Finally, oxidative stress. co-localisation experiments using immunohistochemical tech- Furthermore, we demonstrated a preferential although not niques could help to determine if CNDP2 is a LB component. exclusive CNDP2 overexpression in nigral DA neuron cyto- plasms of PD patients, which might either be a consequence or more interestingly a causative deregulation contributing 5. Conclusions to neurodegenerative processes. First, CNDP2 could be related to oxidative stress. CNDP2 role has not been elucidated but This work offers the largest set of proteins detected so far in the could be linked to carnosine or GSH actions and thus might human SN using a 2-DE proteomic approach. Through careful be crucial to minimize oxidation levels in PD SN and other comparisons between PD and control SN samples, a set of brain parts undergoing degeneration. Thus, CNDP2 increase differentially expressed proteins was established. Among may be viewed either as a compensatory mechanism following them, some tend to confirm the involvement of known path- antioxidant production (GSH, carnosine) in response to oxida- ways in the pathogenesis of PD (i.e. oxidative stress, mitochon- tive stress occurring in PD tissues or as an intrinsic enhancer drial dysfunction), while some others are new candidates of oxidative stress by degrading antioxidant agents (carnosine) eventually pointing toward new mechanisms, such as retromer or perturbing γ-glutamyl cycle. Second, CNDP2 could be linked cargo complex dysfunctions (VPS29). To validate our research to pathological protein aggregation, involving CNDP2 neuronal strategy and to explore one of these original PD-relevant accumulation in toxic oligomers and cellular inclusions. To proteins, we studied in more detail CNDP2, a yet poorly charac- support this hypothesis, the fact that CNDP2 spot identified as terized protein that was found increased in the SN of PD overexpressed in PD patients was slightly shifted compared to patients, as verified by WB analysis. Importantly, CNDP2 was the other constant CNDP2 spot on 2-DE gels. It is known that found to be located in DA neuron cytoplasm by immunohisto- minor changes in protein sequences (i.e. truncation) could chemical analysis, where it might be predominantly increased modify their biochemical properties and structure to promote in PD cases. CNDP2 was also detected in the CSF, suggesting protein misfolding or prevent their degradation, leading to that the protein could be secreted in extracellular fluids. both protein accumulation and aggregation. Third, CNDP2 Altogether, the present study suggests that CNDP2 may be a could be related to inflammation or glial reaction via its secre- new key player in the molecular mechanisms of neuro- tion into the surrounding extracellular fluid. In this case, degeneration underlying PD. CNDP2 role has not been elucidated CNDP2 secreted in the SN extracellular space may become dele- but could be linked to carnosine and GSH actions and thus might terious by modulating its environment and recruiting various be pivotal to control oxidation levels in PD. immune factors or other molecules that could participate to Supplementary materials related to this article can be cell death. Interestingly, a recent study identified CNDP2 as found online at doi:10.1016/j.jprot.2012.02.032. part of metastatic cancer cell secretome and demonstrated its overexpression following hypoxia [37]. To determine whether CNDP2 is secreted in the CSF, Acknowledgments whether it could be measured in it and might eventually be used as a potential, neuropathologically-derived biomarker This work has been made possible through the generosity of of PD, we examined CNDP2 levels in the CSF from PD and the Memorial A. de Rothschild Foundation, the Edmond J. controls patients by WB analysis. No significant differences Safra Philanthropic Foundation, the Gustaaf Hamburger in CNDP2 levels were observed between PD and control groups Foundation, the Ernst and Lucie Schmidheiny Foundation which can be explained by a few hypotheses. 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Chapter V

Human substantia nigra proteomics: insights into Parkinson’s disease pathogenesis

The second experimental part of this thesis project was designed to dig deeper in the substantia nigra (SN) proteome and the specific abnormalities occurring in PD. Human SN post-mortem samples from Parkinson’s disease (PD) and control patients were analyzed by an unbiased high-throughput shotgun proteomic approach combined with isobaric TMT tagging technology for protein quantification. Of note, samples used in the two experimental sections (2-DE and TMT) were mostly different. This study allowed the simultaneous identification and quantification of 1795 proteins, the most extensive catalogue of nigral proteins obtained so far. Moreover, 204 of these proteins displayed significant expression level changes in PD. Some of these findings including Ferritin-L overexpression or seipin underexpression were verified by orthogonal methods. Overall, the complex proteome alterations observed in the SN of PD patients provided further insights into the underlying pathogenic processes engaged in PD.

I performed all the experimental work of this chapter, including data analysis and interpretation. Moreover, I entirely wrote the manuscript that will be submitted to Proteomics.

Chapter V 123

Research Article

Human substantia nigra proteomics: new insights into Parkinson’s disease

Virginie Licker1,2, Enikö Kövari3, Alexander Lobrinus4, Mélanie Côte1,2, Maria Surini-Demiri 3, Denis F. Hochstrasser2, 5, Natacha Turck2, Jean-Charles Sanchez2, Pierre R. Burkhard1, 2, 6

1Neuroproteomics Group, University Medical Center, Faculty of Medicine, 2Translational Biomarker Group, University Medical Center, Faculty of Medicine, 3Department of Psychiatry, Geneva University Hospitals, 4Department of Pathology, Geneva University Hospitals, 5Department of Genetics and Laboratory Medicine, Geneva University Hospitals, 6Department of Neurology, Geneva University Hospitals, Geneva, Switzerland

Correspondence Pierre R. Burkhard, MD

Department of Neurology

Geneva University Hospitals

4, rue Gabrielle-Perret-Gentil

1211 Geneva 14

Switzerland

Phone # +41-22-372 83 09

Fax # +41-22-372 83 32

E-mail address: [email protected]

123

ABSTRACT

Parkinson’s disease (PD) pathology spreads throughout the brain following a highly region- specific process affecting predominantly the substantia nigra pars compacta (SN) since an early phase. SN exhibits a progressive and irreversible loss of nigro-striatal dopaminergic neurons along with the occurrence of Lewy bodies (LB). The resulting deficit in striatal dopamine is responsible for the major cardinal motor symptoms of PD. To gain new insights in the SN function and the specific features making it more vulnerable to neurotoxicity in PD, we analyzed the proteome of post-mortem nigral tissues selectively dissected from pathologically confirmed

PD cases (n=3) and age-matched non-neurological controls. Using high-throughput shotgun proteomic technique involving TMT-labeling, multidimensional separation and LTQ-OT MS/MS, we simultaneously identified 1795 protein groups with concomitant quantitative data. To date, this represents the most extensive catalogue of nigral proteins. Of them, 204 displayed significant changes in their relative abundance in PD patients compared to controls, involved in several known pathogenic processes such as cytoskeletal impairment, mitochondrial dysfunction, energy metabolism failure or oxidative stress. We confirmed the differential expression of Ferritin Light chain, Nebulette, Seipin and Gamma Glutamyl hydrolase.

Interestingly, Seipin might play a role in PD pathogenesis through protein misfolding, aggregation and ER-stress leading to cell death. Overall, this study provides further insights into the underlying pathogenic mechanisms at work in the SN of PD patients.

Chapter V 125

Introduction

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the gradual dysfunction of the extrapyramidal motor system and clinically manifested by tremor at rest, rigidity or bradykinesia. The substantia nigra pars compacta (SN) exhibits a progressive and severe loss of midbrain melanized dopaminergic neurons, resulting in striatal dopamine (DA) deficit, which in turn is responsible for most of the motor symptoms [1]. The pathological process underlying PD extends beyond the SN and targets specific central nervous system (CNS) structures following a stereotyped caudo-rostral progression, according to Braak et al. [2].

Alterations are thought to initiate in the dorsal motor nucleus of the vagal nerve in the medulla and the olfactory bulb, ascend into specific pons and midbrain nuclei, to finally reach cortical regions. Susceptible nerve cells exhibit intracellular inclusions termed Lewy bodies (LB), resulting from the abnormal aggregation of proteins including alpha-synuclein (α-SYN) [3]. Along with neuronal damage, a glial reaction is observed, with the release of various inflammatory factors

[4]. The whole neurodegenerative process requires years to reach its full extent within the CNS and is initiated long before the first motor symptoms become detectable - typically when 50-

70% of nigral neurons are already lost [5]. Without any effective neurorestorative or neuroprotective strategy, PD treatment is still limited to symptomatic DA replacement therapy.

The mechanisms underlying PD pathogenesis remain elusive, although a multitude of scenarios have been elaborated. Several lines of evidence indicate that oxidative stress, mitochondrial dysfunction, impaired protein degradation, excitotoxicity, apoptosis or inflammation may participate to the successive events ultimately leading to neuronal death [6-

9]. The selective vulnerability of DA-containing nigral neurons is generally associated to their higher metabolic rate as well as a greater exposure to oxidative insults through their DA, neuromelanin or iron content for example [10]. Interactions of DA nigral cell populations with their environment might also be an important factor, as for example, even within the SN, cells

125 located in calbindin-poor regions have been shown to be more prone to degeneration than the others [11, 12].

Considerable efforts are made to improve our understanding of PD pathology, a prerequisite for the establishment of disease-modifying strategies. Recently, unbiased large- scale proteomic [13-18] and transcriptomic [19-23] profiling studies of brain post-mortem tissues have revealed additional candidates triggering cell death in PD, involved in neurotrophic support, synaptic transmission, oxidative stress or mitochondrial function for example.

Proteomics allows a global characterization of the effector molecules involved in PD pathological process, whereas transcriptomics suffer from the poor correlation between mRNA and protein expression levels. Among the few proteomic studies investigating human autopsy brain tissue, the majority has focused on SN analysis, including ours, while others have studied cortical regions or recently the locus coeuruleus [13, 14, 16, 18, 24], another aminergic brainstem nucleus degenerating massively in PD. Every single study has the potential to reveal new aspects of the disease pathology, owing to variations in the depth and specificities of the proteome covered in PD patients, that themselves exhibit some level of heterogeneity [25].

We hypothesized that elucidation of some of the complex SN proteome alterations may provide a new source of PD-specific therapeutic targets and biomarkers for the treatment and prevention of PD. We performed a high-throughput quantitative profiling of the SN proteome in

PD patients compared to non-neurological controls (n=6), using an isobaric chemical labeling technique termed tandem mass tag (TMT) that allows the multiplexing of six samples. We identified 1795 proteins groups representing so far the largest nigral proteome map ever published. More than 200 proteins were found to be significantly regulated in PD cases versus controls, 21 of them with a fold change over 1.3. Among them, Nebulette, Seipin, gamma- glutamyl hydrolase and Ferritin Light chain were confirmed by WB or IHC and may be interesting candidates potentially involved in PD pathogenesis. Chapter V 127

Material and methods

1. Human brain tissues

Human midbrain tissues were obtained from the Division of Clinical Pathology of the

Geneva University Hospitals under a procedure approved by the local ethical committee and either frozen at -80°C or fixed at 4°C in 15% formaldehyde for 4 weeks before being paraffin embedded (Table 1). Written consents for brain autopsy and use for research purposes were signed by close relatives. PD cases were clinically diagnosed according to the UKPDSBB criteria and under symptomatic PD treatment before death, except patient P5 considered as an incidental case. PD diagnosis was confirmed neuropathologically by the observation of a severe nigral depigmentation due to neuronal loss and the presence of α-SYN immunoreactive inclusions (LB). P5 may represent an early preclinical stage of PD [26], defined by the absence of symptomatic parkinsonism but the presence of LB in the SN [27]. Controls were cases without nigral abnormalities and with no previous history of neurological or psychiatric disorders.

SN protein extracts were prepared as described in a previously published protocol [17]. Briefly, frozen tissues were cryo-sectioned in 18 μm-thick slices and the SNpc - macroscopically recognizable by its dark pigmentation, was selectively dissected from slides with a scalpel. One mg of nigral tissue per sample was collected and kept at -80°C until analysis.

2. Quantitative sixplex TMT analysis

2.1. Sample preparation and TMT Labeling

Three PD and three control cases (Table 1) were selected for quantitative proteomic comparisons using TMT labeling technology. Groups were matched for patients’ age (mean ± standard deviation of 72.7 ± 13.2 years for Control and 79 ± 6 for Parkinson patients) and post- mortem interval (Mean ± standard deviation of 24 ± 11 years for Control and 18.7 ± 10.1 for

Parkinson patients) (<38h) (Table 1).

127

One mg of each patient’s tissue was suspended in 150 μL of sample buffer containing 6M Urea,

0.1M TEAB (pH 8.0), 0.1% CHAPS, 0.05% SDS, 0.2mM PMSF, phosphatase inhibitors (1mM NaF,

1mM Na3VO4) and a protease inhibitor mixture (Roche). The suspensions were vortexed for 60 min at 4°C, sonicated gently and centrifuged at 4°C, 13’000 x g for 5 min to remove cellular debris. Protein concentrations were determined by Bradford assay (Protein Assay, Bio-Rad,

Hercules, CA).

Fifty micrograms of proteins from each of the six samples were analyzed as described by

Dayon et al [28] with slight modifications. Equal amounts (1 μg) of bovine β-lactoglobulin (LACB) were spiked in each sample as an internal standard to correct for eventual experimental bias.

Briefly, protein reduction was performed in 2.5mM tris-(2-carboxyethyl) phosphine hydrochloride for 60 min at 37°C followed by alkylation in 10mM iodoacetamide for 30 min in the dark at room temperature. Samples were diluted three times to reduce urea concentration for trypsin digestion (trypsin:protein ratio 1:25) that was performed overnight at 37°C. The resulting peptide mixtures were tagged with the sixplex TMT reagents (Thermo Scientific,

Rockford, IL, USA)). The control samples C1, C2, C3 were labeled with TMTs 126.1, 128.1, and

130.1 and the PD samples P1, P2, P3 with TMTs 127.1, 129.1, and 131.1, respectively. After one hour incubation, the labeling reactions were stopped with hydroxylamine. The six samples were finally pooled and dried for storage at -20°C.

2.2. Off-gel peptide fractionation

Prior to off-gel electrophoresis (OGE), the mixture of TMT6 labeled peptides was reconstituted in 150 µl of 5% ACN/0.1% trifluoroacetic acid (TFA) and purified with a C18 macrospin column (Harvard Apparatus) according to the manufacturer instructions. Peptides were recovered after elution with 400 μL of 50% ACN/0.1% TFA and evaporated to dryness. Chapter V 129

Peptides were then fractionated in-solution on the basis of their isoelectric point (pI) with an Agilent 3100 OFFGEL fractionator (Agilent Technologies) according to the manufacturer's protocol (Agilent). Briefly, the samples were dissolved in OFFGEL solution containing 6% glycerol, 0.75% IPG buffer pH 3-10 (GE Healthcare). Isoelectric focusing was done on a commercial 24 cm IPG pH 3−10 linear dry strip (GE Healthcare) with a 24 well frame set, until

50kV/h was reached with a maximum current of 50 μA and power of 200mW. Peptidic fractions were collected and pHs were measured to check for the accuracy of the gradient. Fractions were purified using C18 microspin column (Harvard Apparatus) according to the manufacturer instructions. Elution was performed with 300 µl 50% ACN, 0.1% formic acid (FA). Eluates were evaporated and stored at −20 °C prior MS analysis.

2.3. LC-MS/MS analysis

Each peptidic fraction was reconstituted in 5% ACN/0.1% FA and a third of it was loaded for liquid chromatography LC−MS/MS analysis on a LTQ Orbitrap XL mass spectrometer (Thermo

Electron, San Jose, CA) equipped with a NanoAcquity HPLC system (Waters Corporation, Milford,

MA). Each sample was injected twice on two different days to obtain technical replicates and to increase protein coverage. Peptide separation and MS analysis was performed as described elsewhere [29]. Briefly, peptides were separated for 30 min on a home-made analytical column using a gradient of 99.9% H2O/0.1% FA (solvent A) and FA 99.9% ACN:0.1% FA (solvent B). To obtain precise peptide quantification without compromising peptide identification, ESI LTQ-OT

MS analysis combined collision induced dissociation (CID) and high energy C-trap dissociation

(HCD) activation modes in the LTQ-OT. MS survey scans were acquired in the OT analyzer within an m/z window from 400 to 2000. A maximum of three precursor ions were selected for parallel fragmentation in CID or HCD and subsequent MS/MS detection in the LTQ or OT, respectively.

129

2.4. Peptide and protein identification

Peak lists were generated from raw data using EasyProtConv, a new tool described in

Glück et al (submitted article). Briefly, the tool allowed merging CID and HCD spectra for simultaneous identification and quantification [29]. The 24 OGE fractions from the first and second injections were merged into either separate files to compare between replicates or a unique file. Data were then submitted to Easyprot, a java-based web platform which uses

Phenyx (GeneBio, Geneva, Switzerland) for protein identification (Glück et al, submitted article).

Searches were conducted against UniProt Swiss-Prot database (2011_02 of 08-Feb-2011) alternatively specifying Homo sapiens or Bos taurus taxonomy to search for the spiked LACB.

Trypsin was selected as the proteolytic enzyme, one missed cleavage was allowed, cysteines carbamidomethylation, TMT6 amino terminus and TMT6 lysine (+229.1629 Da) were set as a fixed modification whereas oxidized methionine as variable. The minimum peptide length was five amino acids and precursor error tolerance was 10 ppm. False positive ratios were estimated using a reverse decoy database [30]. The dataset was searched once in the forward and once in the reverse database. Peptide z-scores were then set to maintain a false positive peptide ratio below 1%. Proteins with at least two distinct peptide sequences were selected. Proteins were grouped based on shared peptides [31] and only protein group reporters (proteins containing all the peptides) were considered in the final output.

2.5. Protein relative quantification

We used the publically-available software package Isobar to calculate protein ratios and use the statistical guidance offered to select differentially regulated proteins between sample classes [32]. TMT6 reporter ion intensities were extracted, corrected for isotopic impurities as provided by the manufacturer, and each channel was normalized imposing equal median intensity. Only spectra from specific peptides that were not eliminated after outlier filtering Chapter V 131

were used for quantification. Protein ratio was obtained by combining the ratios between all pairs of distinct classes (126, 128, 130 vs. 127, 129, 131) measured from its peptide MS/MS spectra. Isobar implements an approach providing a statistical signal noise model that accounts for heterodasticity in the OT, and a biological variability model. For each protein ratio, p-values were computed, that model technical (ratio p-value) and biological variability (sample p-value) to estimate ratio accuracy and biological significance, respectively. Biological sample P-values were estimated on the basis of the random ratio distribution (Cauchy model) that was built using intraclass pairwise comparisons. We set the level of risk at 5%, requiring both p-values to be lower for differential expression selection. Isobar also addresses the problem of opposite direction ratios, by taking it into consideration in the overall protein ratio and sample p-value calculation. All details and mathematical demonstration are provided in Breitwieser et al [32].

3. Gene ontology analysis

3.1. Total SN proteome dataset

First, identified proteins were classified by protein class through PANTHER (Protein

ANalysis THrough Evolutionary Relationships) online tool v7.2 [33]. The protein distribution in subcellular localization and biological processes was analyzed using Gene Ontology (GO) Cellular

Component (CC) and Biological Process (BP) via DAVID web-based tool (Database for Annotation and Integrated Discovery) v6.7 ([34, 35].

For the enrichment analysis of Gene Ontology GO Biological process (BP), we used

BINGO v2.0 plugin [36] from Cytoscape v2.6 [37] to obtain statistically over-represented annotations. We compared our nigral dataset with the default human reference dataset containing all GO annotations taxonomy, with p-values computed using the hypergeometric statistical test. Significant GO terms with p-value lower than 0.001 after multiple testing correction with Benjamini and Hochberg false discovery rate and with at least five associated proteins were selected. Of note, gene name correspondence for each identified protein group

131 was used, as listed in Supplemental file 1. Fold enrichment for the i overrepresented BP term of the set BP[i], was calculated as follows: % proteins annotated with BP[i] among the annotated proteins of SN dataset divided by the % of proteins annotated with BP[i] among the annotated proteins of human reference dataset.

KEGG pathway enrichment analysis was performed using DAVID by comparing the nigral dataset against a reference default dataset of all the human annotated proteins. Enriched KEGG pathways were determined using modified Fisher's exact tests and significant KEGG pathways with p-value lower than 0.05 after Benjamini correction. Categories with a minimum of 5 proteins were selected. Fold enrichment was calculated following the same reasoning as for GO

BP. Chapter V 133

3.2. SN differential dataset

For the SN differential protein dataset, functional classification was performed using

Panther. Enrichment in CC and BP GO was performed using DAVID, with p-value lower than 0.05 after Benjamini correction. Fold enrichment were calculated following the same reasoning as explained above. For both analyses, a minimum count of 5 proteins per category was required.

4. Western Blotting verification

The expression level of the selected proteins Ferritin Light chain (FRIL) and Seipin

(BSCL2) was assessed by western blot on autopsy SN tissue of four Control and four PD patients, including cases (C1, C3 and P1, P3) of our proteomic analysis (see Table 1). Groups were matched for patients’ age (mean ± standard deviation of 83 ± 8.8 years for Control and 79 ± 6.4 for PD patients) and post-mortem interval (Mean ± standard deviation of 25.5 ± 9.7 years for

Control and 28 ± 6.7 for Parkinson patients) (<35h) (Table 1). All samples were prepared as for the TMT analysis, see above in section 2.1. Ten micrograms of nigral proteins from each sample

(C1, C3, C4, C5 and P1, P3, P4, P5) were separated on a 12.5% SDS-PAGE and blotted on a nitrocellulose membrane. Blots were probed with rabbit polyclonal to FRIL (1:4000, ab69090,

Abcam), rabbit polyclonal to seipin (1:2000, ab106793, Abcam) and mouse monoclonal to actin

(1:30’000, Sigma). Anti-rabbit and anti-mouse horseradish peroxidase conjugated secondary antibodies (Dako) were diluted at 1:2000. Detection of immunoreactive bands was performed using the chemiluminescent ECL Western Blotting System (GE Healthcare) and quantification of band volumes with TotalLab Quant (Non Linear Dynamics). Actin was used as a reference protein for normalization. Statistical analysis was performed using the two-tailed Mann-Whitney t-test.

133

5. Immunohistochemical analysis

Immunohistochemical staining of three selected proteins FRIL, Gamma glutamyl hydrolase (GGH) and Nebulette (Nebl) was performed on paraffin-embedded SN tissues of a

Control (C6) and a PD (P6) patient. Sections were cut at 12 µm before being deparaffinized in xylene and dehydrated in ethanol (60°). After microwave-induced antigen retrieval in citrate buffer, sections were incubated with 0.25% potassium permanganate in PBS followed by 1% potassium bisulfite/1% oxalic acid to bleach neuromelanin pigmentation. Then, sections were hybridized overnight at 4°C with rabbit polyclonal to FRIL (1:500, ab69090, Abcam), rabbit polyclonal to GGH (1:200, HPA025226, Sigma) or goat polyclonal to Nebl (1:200, NBP1-45223,

Novus Biologicals) diluted in 0.3% Triton-X/1% BSA in PBS followed by 1h at room temperature with anti-rabbit or anti-goat horseradish peroxidase conjugated secondary antibodies (Dako), diluted in 0.3% Triton-X/1% BSA in PBS. Staining was visualized using 3, 3’-diaminobenzidine

(DAB, Sigma) as a chromogen and sections were counterstained with Cresyl Violet. No staining was detected on negative controls, treated identically with hybridization wit primary antibody omitted.

Chapter V 135

Results and discussion

1. Protein identifications in the SN proteome

SN autopsy tissues were selectively dissected from PD (n=3) and control (n=3) patients before being submitted to an unbiased shotgun proteomic technique involving TMT-labeling, which allowed the simultaneous identification and quantification of their proteome. To reduce sample complexity, labeled peptides were separated in 24 Offgel fractions, each of them being analyzed by RP-LC coupled with LTQ-OT MS/MS. From a total of 10’809 unique peptides, 1795 protein groups were identified with at least two distinct peptides. Proteins with at least one specific reporter peptide were counted as identified. Entries with shared peptides - such as isoforms – indistinguishable by MS were clustered in the same protein group, with the protein containing the most peptides selected as the group reporter. All identified protein groups are listed in Supporting file 1, with details on peptide MS identification and quantification. Of note, an overlap of 1183 protein group identifications (67%) was found between the two injections.

Until now, five other publications have explored the SN proteome of PD patients [13-15,

17, 18]. Of them, that by Kitsou et al. could not be considered for further comparison, due to SN missing dataset[15]. Three studies used 2-DE technology yielding a total of 191 protein identifications [13, 17, 18], whereas Jin et al. analysis by multidimensional LC-MS/MS allowed the identification 842 protein groups [14]. We compared protein groups identified in the current study with the above mentioned using UniprotKB Accession Numbers (AC) (details can be found in Supporting file 2). For comparison with Jin et al., the 1373 IPI numbers of the 842 identified protein groups were mapped to corresponding 2016 Uniprot AC using UniprotKB ID mapping option (http://www.uniprot.org/). Of note, 32% (446) of the IPI numbers could not be mapped to any UniprotKB AC mainly due to their removal from IPI database or a change in their IPI number preventing their automatic retrieval. Overall, the comparison revealed that about 33 % of our protein identifications overlap with those of other studies. A high overlap (i.e. 87%) was

135 observed for proteins identified in 2-DE studies, which suffered from a limited dynamic range. Of note, Jin et al. dataset was enriched in mitochondrial proteins and cysteine-containing proteins through the use of ICAT quantification technology, inducing a bias which could account for the smaller overlap (about 60% of protein identifications) with our dataset.

To date, our workflow enabled the identification of the highest number of protein groups (1795) with concomitant quantitative data. Moreover, about 1200 proteins of our newly delineated nigral proteome dataset were not described in the previous SN proteome analyses

(listed in Supporting file 2).

2. SN proteome characterization

2.1. Functional classification and cellular localization

In order to get more information on the identified nigal proteomic dataset overall composition, we performed a PANTHER functional class and Gene Ontology (GO) cellular components analyses. All 1795 identified nigral proteins were first categorized using PANTHER classification and represented in Figure 1. The major functional protein classes were hydrolase

(11.2%), enzyme modulator (8.8%), (8.4%), transferase (8.1%) and cytoskeletal proteins (8%). A small number of proteins were classified as transcription factors, defense/immunity proteins, or extracellular matrix proteins.

Using cellular component (CC) vocabulary, a gene ontology (GO) analysis of the protein groups was performed as illustrated in Figure 2. The largest proportion (82%) of proteins was annotated as cytoplasmic (Figure 2A), with a preferential (>50%) distribution in “cytosol”,

“mitochondrion” or “cytoskeleton” (Figure 2B) structures. Interestingly, 3.5% of proteins were localized in “melanosome”, defined as an intracellular organelle specialized in melanin synthesis and storage, a specific feature of the SN. Neuromelanin, a brain DA-derived pigment typically found in dopaminergic neurons, accounts for the SN characteristic dark-brown color. The importance of melanosomes and neuromelanin in nigral tissue is highlighted by the fact that Chapter V 137

pigmented neurons were shown to be more prone to oxidative damage and cell death, suggesting an involvement of these structures in PD pathogenesis.

About 11% (193 over 1686) of the annotated proteins were parsed into GO terms specific to neurons, illustrating the ability of our approach to detect not only proteins from the major glial cell types (>90% cells) but also from the scarcer neuronal cells (<10% ) in the SN autopsy tissue (Figure 2C). Proteins were associated with different neuronal compartments including “synapse” (6%), “axon” (4%) or “neuronal cell body“ (3%), reflecting the complex morphology and the highly polarized character of the neurons, which need to maintain molecularly and functionally distinct compartments to ensure proper neurotransmission. Some proteins (2%) were associated to “synaptosome”, defined as the structure preserved after tissue homogenization formed by isolated nerve terminals including presynaptic, post-synaptic membrane and density. This structure is commonly used to study synaptic function and the ability to isolate and observe molecular changes at this level is of particular interest to study PD pathogenesis.

2.2. Enrichment in GO biological processes (BP) and KEGG pathways

Then, we performed an enrichment analysis of GO Biological processes and

KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways to get more insights on the SN specific features and processes occurring in the tissue. The identified nigral proteins were clustered based on GO Biological Process (BP) vocabulary using Cytoscape, and KEGG pathways using DAVID. Then, they were compared to the complete human proteome annotations to assess enrichment of significantly over-represented GO categories and KEGG pathways. The same analysis was performed using a dataset approximating the human “central” proteome (CP) which comprised 1124 proteins ubiquitously expressed and experimentally identified in seven human non-neuronal cells lines [38]. Overrepresented GO categories or pathways found in

137 common between the two sets were discarded in order to highlight the processes occurring more specifically in the nigral tissue and remove the influence of the housekeeping proteome

[39].

For GO Biological Process (BP) enrichment analysis, 323 terms were found to be over- represented (p<0.001). 170 of them were found in both SN and CP datasets, broadly covering vital cell biological processes (primary metabolism, proteostasis or cell cycle), and eliminated. Of note, 6 out of 8 over-represented terms exhibiting higher fold enrichment value in the SN than

CP dataset were linked to mitochondrial function and oxidative phosphorylation (see Table 2a).

The remaining 173 GO terms specifically enriched in the SN are listed in supporting file 3, with a selection of them shown in Table 2b. A large portion (31 terms), covering 20 % of the annotated proteins, was related to neuronal activities such as synaptic transmission (i.e. G0:0001505: 4.2), plasticity (i.e. GO:0048167: 3.2), organization (i.e. GO:0050808: 4.9) or development (i.e.

GO:0050770: 2.6). Many terms were linked to the generation of ATP, through glycolysis (i.e.

GO:0005975:1.6), TCA cycle (i.e. GO:0006102:8.7) and oxidative phosphorylation (i.e.

GO:006120:5.7). Other main enriched categories included cytoskeleton organization (i.e.

GO:0030866:6.2), regulation (i.e. GO:0008064:3.9) and transport (i.e. GO:0010970: 5.1), fatty acid (i.e. GO:0006635: 3.3) and amino acid (i.e. GO:0009065: 5.5) metabolisms or response to oxidative stress (i.e. GO:0034599:2.8).

For KEGG pathway enrichment analysis, 39 pathways were found to be over- represented (p<0.05). Fifteen KEGG pathways were found in common between both SN and CP datasets and discarded. The 24 remaining significant over-represented pathways are shown in

Table 3 and classified according to their fold enrichment value. Six of the top-enriched pathways

(hsa00280: 4.2, hsa00290: 3.6, hsa00410: 3.4, hsa00250: 2.6, hsa00380: 2.4, hsa00330: 2.4) were related to amino-acid metabolism. Fatty acid (hsa00062: 4.3, hsa00071: 3.4) and carbohydrate metabolic (hsa00650: 3.4, hsa00051: 2.7) pathways were also shown to be enriched. “Long-term depression” (hsa04730: 2) and “Long term potentiation” (hsa04720: 2.3) Chapter V 139

were directly linked to neuronal activities and synaptic transmission. “ contraction” (hsa04260:2.4) which unexpectedly showed up, comprised mainly proteins involved in Ca2+ signaling pathways and mitochondrial oxidative phosphorylation. Enrichment in Ca2+ signaling pathway (hsa04020:1.5), actin cytoskeleton regulation (hsa04810:1.5), endocytosis

(hsa04144: 1.5) or cell-to-cell communication (hsa04540: 2.3, hsa04530:1.8, hsa04520: 1.7) pathways was also observed. Of note, KEGG analysis also detected less conventional pathways, linked to disease or immune response as well as oocyte meisosis, which in fact included proteins related to general processes or signaling pathways.

Altogether, this analysis supports the notion that SN function requires high energetic supply as suggested by the enrichment in many terms and pathways linked to ATP production.

Correct SN function and neurotransmission in particular might rely on proper vesicle transport and trafficking, protein homeostasis or signaling. The pivotal role of effective antioxidant defense mechanisms in the SN - where oxidative insult is known to be increased, is substantiated by the enrichment in terms linked to oxidative stress response. Over-represented terms and pathways related to amino acid and lipid metabolisms emphasized their biological importance in the brain. Amino acids are known to be enriched in nervous tissues where they participate to the synthesis of many biologically active molecules including proteins, lipids or hormones, can serve as a source of energy through links with TCA cycle and assume specific roles as neurotransmitters or neuromodulators. The SN, like brain tissues in general, exhibits a highest concentration of lipids which serve as an energy reservoir -together with co-enriched carbohydrate metabolic pathway, and second messengers in extra- and intra-cellular signaling to modulate apoptosis, inflammation or synaptic activities [40]. Finally, enrichment in Ca2+ signaling pathway, engaged in the regulation of various processes ranging from neuronal activities to apoptosis, highlights its particular importance in the SN. Nigral DA neurons have an atypical physiological phenotype, with an autonomous pacemaker activity relying on voltage dependant

L-type Ca2+ channels. The resulting sustained Ca2+ entry might be responsible for increased and

139 selective DA neuron vulnerability to mitochondrial oxidant stress as well as compromising Ca2+ homeostasis [41]. Overall, any impairment in one or more of the above mentioned processes and pathways might be critical for SN functioning and lead to cell death.

3. Quantitative MS analysis of protein profiles using sixplex TMT

3.1. Differential protein expression levels in PD patients

TMT6-labeling technology was used to compare the nigral proteome of three PD and three non neurological control patients. Simultaneous protein relative quantification of the six samples was obtained through the inspection of the TMT reporter region in MS/MS spectra.

Peptides assigned to multiple protein hits were not considered for quantification and 1794 of the 1795 identified protein groups could be quantified with at least one spectrum. Supporting file 1 contains all details about the quantified protein groups, including peptide reporter intensities, calculated protein ratios and corresponding p-values.

Spiked-LACB quantification was checked for quality control. For each LACB identified peptide, reporter intensities were normalized by the sum of all the reporter intensities in order to determine the relative abundance of each reporter. The standard deviation of LACB reporter relative intensities was calculated for each TMT channel and their mean (i.e 9.9%) was found to be in accordance with the isobaric tagging technique performances [28]. The mean absolute error with respect to the expected theoretical value (1/6) was small, around 2%. As no particular experimental bias was observed, no data normalization according to LACB was applied.

Normalization was performed by imposing equal median intensity in each TMT channel.

Isobar package was used to calculate protein group ratios. The geometric mean of all

1794 protein ratios was close to 1 (i.e 0.987) with about 95% of the ratios falling in a sharp range between 0.8 and 1.25. Thus, little inter-group variability between PD and controls was observed for the majority of the quantified proteins, suggesting relatively equivalent samples in their Chapter V 141

detected protein composition. Moreover, almost all observed ratio (1790 of 1794) values were less than 2 fold change. This could result from the dilution of the signal when it comes from a restricted part of a cell population or a specific cell type. For example, signal from DA neurons, representing about 10% of the cells in nigral tissue, would be expected to be diluted 10 times. In addition, sensitivity issues might prevent the identification and quantification of less abundant differential proteins.

Isobar was used to select proteins whose ratios were measured with a sufficient signal

(ratio p-value <0.05) ensuring a good accuracy and that were significantly regulated between the two groups (sample p-value <0.05). The Cauchy distribution used to estimate biological sample variability based on three replicates (see quality control in supporting file 4) exhibited a relatively sharp distribution of random protein ratios, indicative of little intra-group variability.

This translated into low ratio significance thresholds (<0.91 and > 1.08). Thus, 204 significantly differential proteins were selected and listed in Supporting file 1 (tab 4), matching the above criteria with keratins being excluded from the analysis. Of them, 96 were over-expressed and

108 under-expressed in the SN of PD patients.

3.2. Pathogenic processes in the SN of PD patients

To gain more insights about the nature (function, localization) of the 204 differentially expressed nigral proteins and the specific processes significantly modulated in PD pathogenesis, we performed a functional class and GO analysis. First, the dataset was categorized using PANTHER, with more than 80% of the proteins annotated. The major protein classes were oxidoreductase (representing 15% of the total annotation hits), cytoskeletal protein

(13%) , hydrolase (13%), transferase (10%), nucleic acid binding (9%) but also chaperone (5%) and many terms related to translation (for details, see supporting file 5). Proteins related to oxidoreductase, cytoskeletal protein and chaperone classes, had a tendency to be over-

141 expressed in PD, whereas proteins in the other cited classes seemed to be more prone to under- expression in PD. Then, the dataset was submitted to a GO enrichment analysis of CC and BP using DAVID, by comparison to the complete human protein annotations. GO categories more specifically involved in PD pathogenesis were expected to contain a highest proportion of differential proteins than the average observed in the total human proteome. CC and BP GO annotations were available for 91% and 89% of the proteins, respectively. In a first step, selected enriched GO categories were classified according to their fold change. In a second step, information about protein ratios was mapped to show up or down regulation of proteins in each category. Sixty five GO CC terms were found to be over-represented matching our criteria, with a selection shown in Figure 3-A as well as twenty-two GO BP categories, as shown in Figure 3-B.1

(see complete list in Supplemental file 5). Some more selected terms are represented in Figure

3-B.2, potentially interesting regarding PD pathogenesis although not significant after Benjamini correction.

Both GO CC and BP analyses converged to several pathways previously known to be associated with PD pathogenesis. A role for cytoskeleton in PD pathogenesis was suggested, with potential impairments in its regulation and assembly. Supporting this hypothesis, enrichment and dysregulation in the expression of proteins associated to “cytoskeleton” and “regulation of cell component organization” GO categories were observed. In the CNS, cytoskeletal proteins represent up to a third of brain proteins, playing crucial roles and ultimately ensuring correct neurotransmission (i.e. axon/neurite outgrowth, vesicular transport, exocytosis etc.). We found a predominant over-expression of cytoskeletal proteins in PD patients, which could result from their accumulation or aggregation due to protein conformation alteration (i.e. truncation, mutation, PTM). Consistent with this, cytoskeletal proteins such as neurofilament subunits (NF-L,

NF-M, NF-H) [42] or Microtubule-Associated Protein 2 [43] are known constituents of LB.

Moreover, many PD-linked proteins (i.e parkin, LRRK2, α-SYN) were shown to modulate cytoskeleton acting on microtubule (MT) stability for example [44-46]. Interestingly, we also Chapter V 143

observed an under-expression of proteins involved in intracellular transport processes (i.e.

“vesicle”, “vesicle-mediated transport”, “transport regulation”) which might result from cytoskeletal defects. Thus, protein accumulation and/or neuronal inclusions may contribute to cytoskeleton collapse causing failure of neuronal transport and synaptic signaling, ultimately leading to neuronal death.

The analysis also highlighted a possible impaired cellular energy metabolism and mitochondrial dysfunction in the SN of PD patients, with enrichment in proteins found in

“carbohydrates metabolism”, “generation of precursor metabolites and energy process” as well as “mitochondrial compartments” categories. Those proteins, involved in glycolysis or various mitochondrial functions such as TCA cycle and oxidative phosphorylation, had a predominant decreased expression in PD, pointing toward a reduction of energy metabolism and ATP depletion. Of note, proteins linked to carbohydrate metabolism were partially upregulated. This might represent compensatory mechanisms to counteract mitochondrial dysfunction and to preserve energy, through the pentose phosphate pathway for example. The observed decrease in mitochondrial protein expression could result from enhanced mitophagy (autophagy of mitochondria), in a cellular attempt to remove damaged mitochondria for instance. It could also be pathogenic by itself whith depletion in key proteins preventing correct mitochondrial function. This is consistent with earlier observations, such as mitochondrial complex I activity decrease specifically described in the SN of PD patients [47]. Interestingly, “oxidation- reduction” process was enriched in our differential dataset, with proteins being equally over- or under-expressed. PANTHER class analysis also indicated a large number of .

Altogether, those observations could reflect increased oxidative insult either consecutive to mitochondrial alterations or responsible for them through protein oxidation.

We noticed a decreased expression of proteins located in enriched neuronal compartments such as “synapse” and “neuronal projection”, “synaptic vesicle” or

“melanososme” as well as those involved in “transmission of nerve impulse” , probably resulting

143 from neuronal loss in the SN. Interestingly, “anti-apoptosis” category was enriched in over- expressed differential proteins, which might reflect compensatory mechanisms attempting to promote neuronal survival.

Moreover, enrichment was seen for other interesting terms such as “fatty acid metabolism” and “amino-acid and derivative metabolism”, both maintaining essential functions in the brain and the SN. Protein deregulation in those categories might lead to energy depletion in SN, as they can supply TCA cycle. In addition, fatty acids are known to modulate various signaling pathway related to inflammation or apoptosis, critical for PD pathogenesis. Of note, the observed under-expression of proteins linked to amino acid metabolism could result from neuronal loss associated to a decrease in levels of neurotransmitter.

Finally, the analysis also highlighted potential novel pathogenic hypothesis. For instance, we observed a deregulation of translational process in the SN of PD patients, with enrichment and under- expression of “ribosomal” proteins and “translational elongation” factors.

Interestingly, decrease in protein translation could be due to activation of the unfolded protein response (UPR), a protective mechanism triggered by elevated levels of misfolded proteins that was recently described in PD patient brains [48]. Consistent with this, we observed enrichment in “endoplasmic reticulum” proteins, with deregulated protein expression which could initiate

UPR. Recently, protein synthesis repression has been associated with synaptic dysfunction and neuronal loss in a prion-diseased mice model [49], providing a link to neurodegeneration.

Chapter V 145

3.3. Comparison with other differential proteins found in other proteomic publications

3.3.1. SN datasets

In a next step, we compared our differential nigral dataset with earlier studies profiling

the nigral proteome of PD patients [13, 14, 17, 18], as summarized in Table 4. Data from the

three 2-DE studies were combined in a unique dataset of 41 differential proteins. Swissprot AC

from 119 IPIs differential identifications from Jin et al. [14] were retrieved using Uniprot ID

mapping and EBI DBfetch IPIHistory. Six IPI entries were removed, corresponding to multiple

protein hits or discarded IPI entries. No differential proteins were found in common between the

five studies.

All 41 differential 2-DE spots were identified in our SN proteome but only 8 of these

were significantly differentially expressed in our dataset as well. Many methodological

parameters could explain these differences, with the most striking being the fact that 2-DE

technology can pick up protein isoform changes that can differ from overall protein expression

detected using shotgun technology. All ratios were concordant between the studies, with under-

expression of ornithine aminotransferase as well as over-expression of 14 kDa phosphohistidine

phosphatase, Ferritin-light chain, or Glutathione S transferase P in the SN of PD patients.

Consistent with our previous 2-DE study, Cytosolic non specific dipeptidase protein 2 (CNDP2)

was also found significantly upregulated

Eighty-six of the 119 differential proteins from Jin et al. were found in our SN proteome,

with 15 of them similarly differential in our dataset. Ratios were concordant for the majority of

the proteins such as Neurocan core protein, Electron transfer flavoprotein-ubiquinone

oxidoreductase, or synaptophysin decreased in PD, as well as for peroxiredoxin-1, increased in

PD patients. Ratios were strongly discordant for five proteins including Ig mu chain C region

protein, Tubulin -8 chain, 1-beta, General vesicular transport factor p115 and

Acylphosphatase-2. Discrepancy could be due to several facts. First, Jin et al. compared nigral

145 mitochondrial proteins in PD versus controls, whereas we analyzed the whole extract. Protein expression could differ depending on cellular compartments. For example, the authors demonstrated that mortalin was preferentially decreased in the mitochondrial rather than the cytosolic fraction. Second, Jin et al. pooled their PD (n=5) and control (n=5) samples in two samples, instead of analyzing them individually. As such, they lost biological variation information whereas we took this parameter into consideration for ratio selection to exclude variations induced by a single sample for example. Finally, as PD is a heterogeneous disease, it is possible that depending on the cases, differential proteins vary.

Overall, this comparison delineated key PD pathogenic mechanisms with common differential proteins linked to cytoskeleton impairment (i.e. Co-actosin-like protein, tubulin, moesin, neurocan), oxidative stress (i.e. GSH, CNDP2, Ferritin peroxiredoxin), apoptosis (i.e.

Annexin 5) glial reaction and inflammation (i.e GFAP), energy metabolism and mitochondrial dysfunction (i.e electron transfer F-Q oxidoreductase, pyruvate dehydrogenase), synaptic or vesicular transport impairment (i-e- synaptophysin).

3.3.2. Locus coeruleus (LoC) dataset

Then, proteomic data from the recently published analysis of the pontine nucleus locus coeuruleus (LoC) in patients with PD [50] were compared with altered proteins in our dataset.

The rationale for this comparison includes the following: 1) LoC is known to play a role in PD pathogenesis, with severe neurodegeneration of local noradrenergic neurons occurring earlier in

PD development (Braak stage 2) than SN involvement (Braak stage 3), 2) projections originating from the LoC can modulate DA action of SN [51] and provide an anatomical path for a prion-like propagation of the disease and 3) common neurodegenerative mechanisms might occur in the different PD affected brain regions including LoC and SN, during disease progression. Thus, although LoC alterations are observed in other neurodegenerative diseases, comparison with SN alterations might help to delineate key mechanisms involved in PD. Chapter V 147

Fourty-nine of the 87 proteins found differentially expressed in the LoC of PD patients were seen in our nigral dataset, indicating somehow similarities between proteomes. Eight proteins were found differentially regulated in both SN and LoC studies, as listed in Table 5.

Ratios were concordant and relatively similar for all of them except Galectin-3, a protein involved in glial activation. This discrepancy might reside in the fact that LoC pathology was initiated long before SN, and might have reached a more severe stage at the time of death.

Galectin-3 under-expression in the LoC might trigger a pro-inflammatory response [52], whereas compensatory mechanisms might still be at work in the SN to counteract neuronal loss, with

Galectin-3 over-expression promoting cell survival [53].

Among the other common pathogenic proteins, we found the neuronal protein synapsin whose under-expression in PD might be secondary to synaptic and neuronal loss. We also found over-expressed intermediate filament vimentin strengthening cytoskeletal impairment hypothesis, as well as under-expressed proteins involved in glycosylation (P04843) or G-protein signaling (Q9H2M9) in PD. Interestingly, we observed downregulation of Leucine rich PPR motif containing protein (LRPPRC), whose mutations cause the French-Canadian type of

Leigh syndrome (LSFC), a neurodegenerative disease associated to parkinsonian features. A link with PD was provided by a potential interaction of mitochondrial LRPPRC with PD-associated proteins Parkin and PINK-1 [54, 55]. Together with LRRK2 association to cytochrome c oxidase deficiency [56], those observations offer a potential connection with PD pathogenesis through mitochondrial respiratory chain defect. In addition, we also found downregulation of Seipin, a transmembrane ER protein linked to neurologic disorders (i.e. Silver syndrome, Charcot-Marie-

Tooth syndrome variant or distal hereditary motor neuropathy) and thus potentially important in the CNS [57]. Seipin mutations have been shown to result in conformational changes causing accumulation of misfolded proteins in the ER, UPR pathway activation, ER-Stress and ultimately cell death. Of note, mutations in N-glycosylation motif were shown to enhance seipin ubiquitination and degradation by the UPS, which could account for Seipin decrease in PD

147 patients. These observations suggest a role for seipin in PD pathogenesis through protein aggregation or ER-mediated stress.

4. Verification of differential protein expression levels and association with pathogenic PD hypotheses

4.1. Verification of selected proteins of interest by WB and IHC

We included an additional 1.3-fold change cutoff on all TMT ratios to reduce false positive rates in the selection of differentially expressed proteins for potential verification by WB and/or IHC. The final top-21 differential proteins are listed in Table 5 with potential PD pathogenic hypotheses. We selected both over and underexpressed candidates based on commercial antibody availability and quality. Among them, Seipin, GGH, and Nebulette were chosen as original candidates because no clear link with PD had previously been established. On the other hand, Ferritin-L was also selected because its differential expression in PD brain was still controversial in the literature, with for example a decrease observed in Connor et al. [58] study or no difference found in Faucheux et al.

WB and IHC experiments supported the proteomic findings. WB data demonstrated significant 1.7 fold increase of Ferritin-L and 2.5 fold decrease of Seipin expression levels

(p<0.05, Mann whitney t-test) in the SN of PD patients versus controls (Figure 4). Interestingly, the incidental PD case (P4) exhibited the same pattern of expression as PD patients for both proteins, which may reflect changes occurring early in PD pathogenesis. GGH IHC analysis showed a predominant localization of the protein in DA neuron cell bodies and processes, in the

SN of PD and control patients. The number of GGH immunoreactive neurons in the SN of PD patient was decreased as compared to control, suggesting that reduced expression levels of GGH in PD patients may result from neuronal loss (Figure 5A-B). On the other hand, GGH staining seemed to be more intense in neuronal cell bodies of surviving PD cells, which could indicate a Chapter V 149

potential pathogenic role of the protein. Nebulette immunoreactivity seemed to be confined to

DA neuron cytoplasms, with a marked increased expression observed in PD patients compared to controls (Figure 5C-D). The 1.9 fold change observed in the proteomic analysis might not reflect the extent of over-expression occurring in PD DA neurons, as it might be diluted by signal from the larger number of surviving cells in healthy control tissue. Finally, Ferritin immunoreactivity was predominantly observed in glial cells, in particular those sharing the morphology of oligodendrocytes and microglia (Figure 5E-F). When comparing staining patterns at higher magnification, difference between pathological and healthy states was more obvious, with PD patient exhibiting a more intense nigral staining (data not shown).

4.2 Association with potential PD pathogenic hypotheses

Among the most differential proteins identified in this study (Table 5), we observed altered levels of at least two cytoskeletal proteins, namely Nebulette and Tubulin-8, which were respectively up- and downegulated in PD SN. Nebulette is a member of the family, which participates in the regulation of actin filament structure [59]. While nebulette is thought to be cardiac-specific, the shorter splice variant Lasp-2 (or LIM-nebulette) exhibits a strong expression in brain tissues [60]. Lasp-2 is thought to act as a molecular scaffold for actin but also focal adhesion (FA) stabilization and organization [61]. FAs are structures linking cellular cytoskeleton with extracellular matrix and inappropriate interactions in those regions could lead to neurodegeneration through anoikis programmed cell death [62]. Interestingly, Lasp-2 overexpression in pheocytochroma PC6 cell line was shown to inhibit neurite outgrowth in response to growth factors [61]. Recent data suggested the involvement in PD pathogenesis of actin cytoskeleton remodeling, whose impairment might interfere with various processes including synaptic function [63]. Tubulin α/β heterodimers are microtubule (MT) polymer constituents. A decrease in a tubulin monomer is in line with a disruption of MT dynamics,

149 leading to impairment in vesicular/organelle trafficking and signaling at the neuronal level.

Several lines of evidence have already implicated tubulin and MT in PD [63-65].

Four of our top-ten overexpressed proteins - Ferritin-L, Argininossuccinate synthase

(ASS), AKRC2 and AKRC3 - could be linked to oxidative stress either as a causative factor or an adaptative response to increased oxidative levels observed in PD brains. Alterations in iron- storing ferritins in PD brains have been previously demonstrated in multiple studies [66-68].

Ferritin, made of L and H subunits, exhibits anti-oxidant properties offering protection against iron-mediated radical damage [67]. As demonstrated by IHC analysis, changes in Ferritin-L expression level might arise from glial cell populations, in particular microglia, where the protein is mainly localized. Ferritin-L upregulation in PD might result from the general increase in glial cell populations during inflammation [69] and may be produced for the purpose of binding iron released from dying DA neurons in the extracellular space [70]. The high iron content characterizing nigral dopaminergic neurons might be relevant for their selective vulnerability

[71]. Argininosuccinate synthase (ASS) represents a limiting step in arginine synthesis and nitric oxide (NO) production [72]. Upregulation of ASS could lead to sustained levels of NO, which could induce neurodegeneration through mediation of excitotoxic signaling pathways, damage to DNA or nitration/nitrosylation of proteins [73]. In PD, NO modifies parkin [74] or α- SYN which promotes LB formation when nitrated [75]. Moreover, ASS might be regulated by inflammation as LPS and pro-inflammatory cytokines were shown to trigger increased ASS protein levels [76]. Altogether, this suggest a role for NO and ASS in PD pathogenesis. Aldo-keto reductases (AKR) are known to play central roles in cellular response to oxidative stress with detoxification of reactive aldehydes for example [77]. The observed overexpression of AKR family C2 and C3 members could contribute to the elimination of oxidative stress products.

We observed overexpression of Glycerol-3-phosphate dehydrogenase (GPD1) and

Sorbitol dehydrogenase, two proteins that could be linked to energy metabolism. Overwhelming evidence indicated perturbation in glucose metabolism and mitochondrial respiratory chain in Chapter V 151

PD brains [71], thought to initiate or predispose to neurodegeneration in PD. GPD1 catalyzes the production of glycerol from DHAP and the regeneration of NAD+ for glycolysis. GPD1 also participates to G3P shuttle, which transfers electrons from cytosolic NADH to the mitochondrial electron transport chain, allowing ATP production [78]. Increased GPD1 expression may thus constitute a metabolic adaptation to preserve cellular ATP concentration through alternative processes such as G3P shuttle. Sorbitol dehydrogenase is implicated in the conversion of sorbitol into fructose, which can further enter glycolytic pathway. Increased sorbitol dehydrogenase levels might result in higher substrate levels for glycolysis and increased energy production. Of note, increased concentrations of monosaccharides and energy metabolic intermediates such as fructose could exert neuroprotective properties by fueling glycolysis, as demonstrated in MPP+- induced toxicity cell model [79].

Three of the top-underexpressed proteins in PD (seipin, elongation factor 1 alpha, and endoplasmic reticulum resident protein 29) could be related to protein synthesis and folding. As discussed above, the ER-resident Seipin glycoprotein, has been linked to neurologic disorders

(i.e. Silver syndrome, Charcot-Marie-Tooth syndrome variant or distal hereditary motor neuropathy) [57]. Mutations in seipin were shown to result in protein misfolding, accumulation in the ER, UPR pathway activation, ER-Stress and ultimately cell death. A decreased level of the protein in PD could result from the degradation of misfolded protein forms. ERp29, is a newly discovered ER resident involved in secretory protein synthesis [80]. Elongation factor 1 alpha 2

(eEF1A2) participates in protein translation through the elongation of the polypeptidic chain by aminoacylated-tRNA binding to ribosome. Both Erp29 and eEF1A2 downregulation could indicate a general attenuation in protein synthesis in PD brains under stress conditions.

Interestingly, deletion in Eef1a2 gene in a mice model was shown to abolish its tissue expression, leading to increased apoptosis [81].

Four of the most dysregulated proteins were related to inflammation and glial activation, also thought to participate to DA neuron demise [4]. We, as well as many others

151

(i.e[18]) have found an overexpression of glial fibrillary protein (GFAP) in PD, the principal astrocytic intermediate filament, reflecting the massive astrogliosis occurring in PD SN.

Interestingly, we identified an upregulation of phospho-lipid calcium binding protein annexin A1

(ANXA1) in the SN of PD patient, which might exhibit neuroprotective or anti-neuroinflammatory properties [82]. Although ANXA1 action mechanisms in the brain are still unclear, it could be an important effector in the microglial clearance of apoptotic neurons [83]. We found a decreased expression of Ig mu chain C (IgM), evoking a possible role in humoral immunity in PD. IgM can trigger complement activation pathway, and its decreased expression might result from an attempt to reduce pro-inflammatory signal to protect remaining neurons. The signal regulatory protein-β1 (SIRPβ1), a DAP12-associated transmembrane receptor was found to be downregulated and could induce defect in the counter-regulation of proinflammatory factors

[84] and clearance of aggregated proteins, aggravating PD pathogenesis. A recent study showed that SIRPβ1 knockdown in microglial cells induced impairment in neuronal debris and fibrillary amyloid-β phagocytosis [84].

In addition, we found an overexpression of zonula occludens ZO-2, an endothelial cell tight junction protein. Involvement in PD could be explained by defect in the blood brain barrier which may constitute a causative factor of DA neuron degeneration [85]. Finally, we found dysregulation in proteins for which very few or no functional information was available, such as increased Tetratricopeptide repeat protein 9C and decreased ECM component neurocan core protein, Nodal Modulator and Gamma glutamyl hydrolase. The latter, is a key enzyme in folate metabolism whose decreased expression in PD SN could be due to neuronal loss as assessed by

IHC analysis.

Chapter V 153

5. Conclusions

In this study, we simultaneously identified and quantified the proteome of selectively dissected SN autopsy tissues from PD (n=3) and non-neurological control (n=3) patients, using an unbiased shotgun proteomic workflow involving LTQ-OT MS/MS and TMT labeling.

We were able to delineate the most extensive catalogue of nigral proteins (1795) with corresponding quantitative data. About 60% of the proteins appear to be newly associated to human SN. GO annotations and KEGG pathway analysis suggested that SN function relied on high energetic supply, proper vesicular transport and trafficking, as well as Ca2+ homeostasis, amino acid cycle and anti-oxidant response. SN proteome characterization represents a first step toward the understanding of the SN complexity and specific features, which might hold the key to its vulnerability in PD.

Moreover, we observed significant expression changes in 204 proteins, when comparing the nigral proteomes of PD patients versus controls. GO enrichment analysis indicated that these alterations reflected several known pathogenic processes such as cytoskeletal impairment, mitochondrial dysfunction, energy metabolism failure or oxidative stress as well as potential novel pathogenic hypothesis such as a deregulation of translational process. Some of the candidates had previously been identified by others in the SN (i.e. Ferritin-L, 14kda phosphohistidine phosphatase, CNDP2, GFAP) but also in other affected tissue such as the LoC ( i.e. Synapsin, Seipin). Many novel candidates were emphasized including Nebulette and GGH expressed by DA neurons.

Overall, the complex proteome alterations emphasized in this study provide further insights into the underlying pathogenic processes at work in the SN of PD patients. The elucidation of the precise sequence of events triggering neurodegeneration in PD may ultimately provide new therapeutic targets and biomarkers for the treatment and prevention of PD.

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List of Tables

Table 1. Clinico-pathological data of human post-mortem samples

Table 1. Clinicopathological data of human post mortem samples. Abbreviations: DEM=Parkinson's disease with dementia, DLB=Dementia with LB, DD=disease duration, PMI=post-mortem interval. ID Experiment Diagnosis Gender Age DD PMI C1 TMT-126, WB Ctrl M 70 35 C2 TMT-128 Ctrl F 61 24 C3 TMT-130, WB Ctrl F 87 13 C4 WB Ctrl M 89 23 C5 WB Ctrl M 86 31 Additional patient for IHC (paraffin embedded tissue) C6 IHC Ctrl F 92 34

P1 TMT-127, WB PD M 73 16 25 P2 TMT-129 PD M 79 19 7 P3 TMT-131,WB PD F 85 2 24 P4 WB PD M 84 ? Incidental 38 P5 WB PD M 74 25 Additional patient for IHC (paraffin embedded tissue) P6 IHC PD M 73 16 25

Chapter V 155

Table 2. Gene Ontology Biological Process (GO BP) term enrichment analysis of the nigral proteome. 1626 proteins were linked to at least one annotation a. GO BP terms found significantly over-represented with p<0.001 with a higher fold enrichment in the SN dataset than CP dataset. Differences in fold enrichment values of at least one unit were retained.

fold fold GO ID GO BP enrichment enrichment in in SN CP 22904 respiratory electron transport chain 5.5 3.5 34654 nucleobase, nucleoside, nucleotide and nucleic acid biosynthetic process 5.4 4.2 42773 ATP synthesis coupled electron transport 5.2 3.6 42775 mitochondrial ATP synthesis coupled electron transport 5.2 3.6 6839 mitochondrial transport 4.8 3.8 6119 oxidative phosphorylation 4.7 3.7 22900 electron transport chain 4.6 3.4 32270 positive regulation of cellular protein metabolic process 4.6 2.6

b. Representatives GO BP terms found significantly over-represented in the nigral proteome. For each term, fold enrichment is given, representing the ratio of its frequency in the identified SN proteome dataset compared to the GO annotations of the entire human proteome. BPs related to neuronal activities are represented in bold.

GO ID GO BP term Fold enrichment # Genes 48489 synaptic vesicle transport 5.2 19 50808 synapse organization 4.9 9 1505 regulation of neurotransmitter levels 4.2 33 48167 regulation of synaptic plasticity 3.2 21 50770 regulation of axonogenesis 2.6 19 6102 isocitrate metabolic process 8.7 5 6108 malate metabolic process 6.6 6 50690 regulation of defense response to virus by virus 6.6 6 30866 cortical actin cytoskeleton organization 6.2 10 6120 mitochondrial electron transport, NADH to ubiquinone 5.7 28 51193 regulation of metabolic process 5.6 9 9065 glutamine family amino acid catabolic process 5.5 12 10970 microtubule-based transport 5.1 14 15986 ATP synthesis coupled proton transport 4.2 20 8064 regulation of actin polymerization or depolymerization 3.9 27 7006 mitochondrial membrane organization 3.6 12 43254 regulation of protein complex assembly 3.3 36 6635 fatty acid beta-oxidation 3.3 28 910 cytokinesis 2.9 16 34599 cellular response to oxidative stress 2.8 17 60627 regulation of vesicle-mediated transport 2.6 36 55114 oxidation reduction 2.4 179 7264 small GTPase mediated signal transduction 2.4 79 7568 aging 2.1 35

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Table 3. KEGG pathways found significantly enriched in the nigral proteome. 895 proteins (about 50%) were linked to at least one KEGG pathway. The number of proteins associated with each GO term and associated fold enrichment information are indicated for each GO term.

KEGG-ID KEGG pathway Fold enrichment count hsa00062 Fatty acid elongation in mitochondria 4.3 6 hsa00280 Valine, leucine and isoleucine degradation 4.2 32 hsa00290 Valine, leucine and isoleucine biosynthesis 3.6 7 hsa00071 Fatty acid metabolism 3.4 24 hsa00410 beta-Alanine metabolism 3.4 13 hsa00650 Butanoate metabolism 3.4 20 hsa00051 Fructose and mannose metabolism 2.7 16 hsa00250 Alanine, aspartate and glutamate metabolism 2.6 14 hsa05110 Vibrio cholerae infection 2.5 24 hsa00380 Tryptophan metabolism 2.4 17 hsa04260 Cardiac 2.4 33 hsa00330 Arginine and proline metabolism 2.4 22 hsa04720 Long-term potentiation 2.3 28 hsa04540 Gap junction 2.3 36 hsa05120 Epithelial in Helicobacter pylori infection 2.1 25 hsa04730 Long-term depression 2.0 24 hsa04666 Fc gamma R-mediated phagocytosis 1.9 31 hsa04530 Tight junction 1.8 42 hsa04114 Oocyte meiosis 1.8 34 hsa04520 Adherens junction 1.7 23 hsa04912 GnRH signaling pathway 1.7 29 hsa04810 Regulation of actin cytoskeleton 1.7 63 hsa04144 Endocytosis 1.5 49 hsa04020 Calcium signaling pathway 1.5 45 Chapter V 157

Table 4. Comparison of differential proteins found in the SN of PD patients across proteomic studies. Discordant ratios are shown in italic. a. Common differential proteins between Licker et al. and 2-DE datasets Licker et al. 2-DE a, b, c Swissprot AC protein name PD/C ratio PD/C ratio P04181 Ornithine aminotransferase, mitochondrial 0.91 0.46c Q96KP4 Cytosolic non-specific dipeptidase 1.17 2.47c P08758 Annexin A5 1.19 1.32b Q14019 Coactosin-like protein 1.20 2.02b Q9NRX4 14 kDa phosphohistidine phosphatase 1.24 1.72c P09211 Glutathione S-transferase P 1.25 1.32b P02792 Ferritin light chain 1.57 1.58c P14136 Glial fibrillary acidic protein 1.63 2.59b b. Common differential proteins between Licker et al. and Jin et al. datasets Licker et al. Jin et al, 2006 Swissprot AC protein name PD/C ratio PD/C ratio P04220 Ig mu heavy chain disease protein 0.29 14.29 Q9NY65 Tubulin alpha-8 chain 0.71 10 O14594 Neurocan core protein 0.74 0.70 P24534 Elongation factor 1-beta 0.80 2.70 Q9H598 Vesicular inhibitory amino acid transporter 0.82 0.68 P00367 Glutamate dehydrogenase 1, mitochondrial 0.83 0.80 Q16134 Electron transfer flavoprotein-ubiquinone oxidoreductase, mit 0.84 0.39 O60763 General vesicular transport factor p115 0.86 1.96 P08247 Synaptophysin 0.88 0.71 O00330 Pyruvate dehydrogenase protein X component, mit 0.90 0.20 P26038 Moesin 1.19 5.56 P14621 Acylphosphatase-2 1.20 0.31 Q06830 Peroxiredoxin-1 1.23 1.82 P14550 Alcohol dehydrogenase [NADP(+)] 1.24 999 P05556 Integrin beta-1 1.25 1.67 a b c Basso et al., 2004, Werner et al. 2008, Licker et al., 2012.

Table 5. Common differential proteins found in the SN and LC of PD patients. Table 5. Common differential proteins found in the SN and LC of PD patients SN: Licker LC: Van Dijk, 2011 Swissprot AC protein name PD/C ratio PD/C ratio P08670 Vimentin 1.23 1.37 P17600 Synapsin-1 0.89 0.83 P42704 Leucine-rich PPR motif-containing protein, mit 0.79 0.84 P04843 Dolichyl-diphosphooligosaccharide-protein glycosyltransferase sub 1 0.86 0.52 P17931 Galectin-3 1.24 0 Q9H2M9 Rab3 GTPase-activating protein non-catalytic subunit 0.83 0.4 Q96G97 Seipin 0.67 0.59 O75534 Cold shock domain-containing protein E1 0.83 0.45

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Table 6. Top- differentially expressed proteins in the SN of PD patients versus controls. Differential proteins exhibiting a fold change over 1.3 were ranked based on PD/C ratio calculated using Isobar (sample and ratio p-values <0.05). Associated main PD pathogenic hypotheses were listed, with asterisks representing less conventional or novel pathways.

AC protein name PD/C ratio main PD pathogenic hypothesis Top-overexpressed proteins in the SN of PD patients O76041 Nebulette 1.86 * Cytoskeleton structure/regulation P21695 Glycerol-3-phosphate dehydrogenase [NAD(+)], cyt 1.74 Energy metabolism Q9UDY2 Tight junction protein ZO-2 1.71 * Blood-brain barrier communication P14136 Glial fibrillary acidic protein 1.63 Glial activation and inflammation P04083 Annexin A1 1.58 Glial activation and inflammation P02792 Ferritin light chain 1.57 Oxidative stress P00966 Argininosuccinate synthase 1.53 Oxidative stress (NO), Protein modification P42330 Aldo-keto reductase family 1 member C3 1.49 Oxidative stress Q00796 Sorbitol dehydrogenase 1.46 Energy metabolism P52895 Aldo-keto reductase family 1 member C2 1.44 Oxidative stress Q8N5M4 Tetratricopeptide repeat protein 9C 1.39 unknow n Top-underexpressed proteins in the SN of PD patients P01871 Ig mu chain C region 0.29 Glial activation and inflammation Q92820 Gamma-glutamyl hydrolase 0.64 unknow n O00241 Signal-regulatory protein beta-1 0.66 Glial activation and inflammation Q96G97 Seipin 0.67 Apoptosis P69849 Nodal modulator 3 0.70 Unknow n Q9NY65 Tubulin alpha-8 chain 0.71 * Cytoskeleton structure/regulation Q53FP2 Transmembrane protein 35 0.72 Unknow n Q05639 Elongation factor 1-alpha 2 0.73 Protein synthesis, apoptosis O14594 Neurocan core protein 0.74 * ECM structure/regulation P30040 Endoplasmic reticulum resident protein 29 0.74 Protein folding

Chapter V 159

List of Figures

Figure 1: Functional classification of the nigral proteome. The nigral proteins identified were categorized by protein classes using PANTHER. Functional annotation was available for 1504 of the 1790 proteins. Percentage represents the proportion of annotated protein hits in each category against the total number of hits.

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Figure 2: Subcellular localization of the nigral proteins according to the Gene ontology (GO) annotations of cellular component (CC). GO CC annotations were available for 1686 out of the 1790 proteins. The figure illustrates protein distribution patterns of (A) subcellular localization, (B) cytoplasmic organization or (C) neuronal structure according to selected GO categories. Of note, the sum of percentage exceeds 100% because of multiple annotation terms associated to a single protein.

Chapter V 161

Figure 3. Enriched GO CC and BP categories in the set of differentially expressed nigral proteins. The graph shows significantly (Benjamini correction, p<0.05) over-represented GO CC (A) and GO BP (B.1) categories classified according to their enrichment fold value. Some more GO BP terms potentially interesting regarding PD pathogenesis are shown in B.2. They were significant before Benjamini correction but not after. In the left panel, for each GO term, fold change is given, representing the ratio of its frequency in the differential SN proteome dataset (204 proteins) compared to the GO annotations of the entire human proteome. In the right panel, the number of over-expressed and under-expressed proteins in the SN of PD patients is given for each GO category. To simplify the scheme and minimize redundant information, a representative selection of GO terms is shown in the graph. For the complete list of enriched BP and CC categories, see Supporting file 5.

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Figure 4: Confirmation of proteomic candidates in the SN of PD patients. WB of two selected proteins, Ferritin-L in Panel A and Seipin in Panel B. Bar graphs represent average β actin- normalized band volumes with SD. Ferritin-L level is significantly increased 1.7 fold whereas Seipin is decreased 2.5 fold in PD patients' SN (p < 0.05, unpaired t test).

Chapter V 163

Figure 5. Immunohistochemical localisation of GGH, Nebulette, and Ferritin-L in SN paraffin sections of control (A, C, E) and PD (B, D, F) patients.

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Chapter VI

General discussion, perspectives and conclusion

Chapter VI 171

VI. GENERAL DISCUSSION

Towards the elucidation of Parkinson’s disease pathogenesis

Parkinson's disease (PD) is a common neurodegenerative disorder mainly characterized by motor symptoms caused by to the loss of DA neurons in the substantia nigra pars compacta

(SN). Nigral neurodegeneration occurs in the presence of intraneuronal proteinaceous aggregates termed Lewy bodies (LB) that contain α-synuclein (α-SYN) and other proteins prone to aggregation. Despite decades of intensive research, the precise etiopathological mechanisms underlying sporadic PD remain mysterious, impeding the establishment of disease-modifying or neuroprotective therapeutic strategies. A generally accepted scenario suggests that sporadic PD results from a complex interplay between aging and environment in genetically predisposed individuals. A multitude of cellular and molecular causative hypotheses including α-SYN misfolding and aggregation, oxidative stress, mitochondrial dysfunction or UPS impairment were hitherto shown to contribute to PD neurodegeneration. However, neither unifying nor completely satisfying theory on PD pathogenesis has been established yet. Traditional candidate-based studies focusing on selected biological events have failed to unravel the extremely complex nature of PD processes, which might rather be explored by global unbiased hypothesis-free approaches encompassing “omics” high-throughput technologies. Proteomics has emerged as one of the most attractive strategy to approach PD pathogenesis through the comprehensive analysis of the pathological effectors themselves.

The results presented in this thesis demonstrated that comparative proteomic analyses targeting human SN, the key brain structure selectively affected in PD, were successful in confirming existing theories but also highlighted novel potential pathogenic molecules engaged in PD. Combining two different but complementary proteomic quantitative workflows, 2-DE and shotgun approach using isobaric TMT technology, we established the SN proteome and elucidate

171 some of the complex alterations occurring in PD by comparing the nigral proteomic profiles of

PD versus non-neurological control patients.

1. Using human post-mortem autopsy tissues

In recent years, many proteomic studies were performed in animal models of PD, even overshadowing investigations in human tissues [1]. However, results from these studies may hardly been translated to the human condition, as PD models developed until now cannot recapitulate all clinical and neuropathological features associated with sporadic PD, a typically human specific disease [2, 3]. In consequence, we rather investigated human post-mortem tissues, which represent in our view a unique window into the specific abnormalities occurring in

PD brains [4]. Taken at a late-stage of the pathological process, autopsy tissues can offer an instantaneous view of a constantly evolving disease, although not necessarily reflecting the complex dynamics of the whole degenerative process. These samples offer important advantages including the neuropathological confirmation of PD diagnosis and the possibility to selectively dissect PD-relevant regions such as the SN - or even cellular and subcellular structures

(see Perspectives), to decipher their specific proteome alterations. The rationale for a global whole-tissue approach adopted in this project, was based on the fact that the chosen proteomic strategies could enable the observation of neuronal specific changes and that glial cells were known to contribute to PD pathogenesis as well [5].

The use of autopsy tissues entails its own set of difficulties and limitations that needs to be kept in mind when interpreting data. In contrast to animal or cell models, tissues’ availability is limited in terms of sample type, number, quantity or quality. A general trend towards a dramatic decline in autopsy rates has been observed worldwide [6] with Geneva University

Hospitals not being an exception. This reduction can be explained by i) a reduced autopsy interest from clinicians, ii) the fact that autopsy is no longer automatically performed, iii) patient and family consents getting more difficult to obtain, and iv) autopsy costs, not covered when Chapter VI 173

patients die at home or in nursing homes. Consequently, the limited number of patients’ samples obtained during this thesis did not allow the design of statistically powerful case-control studies regarding sample size. In addition, the locus niger is a small nucleus and the amount of tissue obtained per sample was not sufficient to cover all proteomic experiments. Some samples were thus entirely consumed after 2-DE experiments and different samples were used for the shotgun approach or western blot verification. Tissue quality may vary according to storage conditions and sometimes long post-mortem delays (PMD). We selected samples with PMD under 38h, matched between control and PD groups, in accordance with Crecelius et al. observations stating that the majority of proteins degrade after 48h at room temperature [7].

New samples fulfilling our quality criteria were obtained at a rate of 2 per year approximately.

Patients were carefully selected. PD hallmarks were found in all PD cases and absent in all control cases, as assessed by neuropathological examination of the SN. All PD samples were clinically diagnosed except for a recently acquired one (chapter IV, patient P5, western blot).

This particular sample came from an incidental LB case, defined by the absence of symptomatic parkinsonism but the presence of LB in the SN [8], which might represent an early preclinical stage of PD [9]. Controls had no previous history of neurological or psychiatric disorders.

Because PD is a heterogeneous disease, different causative factors might occur among individuals PD patients presenting with different PD subtypes. Here, patients had a typically late- onset PD with variable disease duration (from 2-19 years). Patient’s co-morbidities, aging and pharmacological treatments also affect protein levels and could account for changes unrelated to disease.

2. Characterizing the substantia nigra proteome

In this project, we overall defined the largest catalogue of nigral proteins with concomitant quantitative data. The gel-based 2-DE approach presented in Chapter IV allowed the MS/MS identification of 257 spots corresponding to 163 proteins. The resulting 2-DE map is

173 the most extensive established so far, with 112 novel protein identifications [10, 11]. In addition, it provides valuable information on nigral protein isoforms (i.e. post-translational modifications,

PTMs), which can be separated on a 2-DE gel owing to variations in their charge (pI) or MW. This

2-DE map represents a reference for further 2-DE studies on SN proteome as it might facilitate the rapid identification of protein alterations when performing analyses with different patients’ samples, or using a DIGE approach for instance. For an easiest access, the 2-DE map will be integrated into SWISS-2DPAGE online database. Although informative and reflecting some aspects of SN proteome complexity, this 2-DE proteome map is nevertheless incomplete. First, less than 20% of the spots detected on the 2-DE gel were identified, mainly due to i) a time- consuming identification process not automated for 2-DE high-throughput analysis, and ii) the detection limits of the mass spectrometer (i.e. MALDI-TOF-TOF) preventing the identification of the faintest spots. Second, some classes of proteins are typically under-represented on 2-DE gels, such as very acidic or basic proteins, hydrophobic or membrane proteins. Third, the dynamic range of protein expression represented on a 2-DE gel is limited, preventing the simultaneous identification of low (down to the nanogram for silver staining) and high abundant proteins.

Accordingly, a second study presented in Chapter V was designed to increase nigral proteome coverage, using a gel free shotgun approach combining OGE fractionation of TMT-6 tagged peptides with highly sensitive LTQ orbitrap for rapid protein identification. 1795 protein groups were identified comprising 149 of the 2-DE proteins and up to 1200 proteins newly associated with human SN [10-13]. For an easiest access, the dataset will be publicly available in

NextProt database, a web-accessible resource providing high quality information on human proteins. GO annotations and KEGG pathway analyses were performed, enabling the further characterization of this large dataset regarding proteins’ cellular localization and biological function. A significant proportion of annotated proteins were parsed into GO categories related to neuronal components (11%) or activities (20%), validating the ability of our whole tissue Chapter VI 175

approach to detect the scarcer neuronal cell populations (5-10%) in the SN tissue. Moreover, overrepresentation analyses suggested an important role for energy metabolic pathways, cytoskeletal organization, proper vesicular transport and trafficking, Ca2+ homeostasis, amino acid cycle, cell-cell junctions (tight, gap, adherens) or anti-oxidant response in the SN. We could infer that any perturbation in one or more of the above cited processes might be particularly critical for SN correct function.

Taking advantage of the 2-DE potential to resolve protein isoforms together with the high-throughput capability of the shotgun approach, we managed to get the most comprehensive picture of the human SN proteome so far. Neuronal degeneration in PD is region-specific, affecting predominantly the brainstem region particularly in its early stage. Thus, establishing SN proteome constitutes a first step towards a better understanding of the SN function and the specific features making it more vulnerable to neurotoxicity in PD. To further address the question of pathology regional selectivity and cellular vulnerability in PD, SN proteome might be compared with those of different brain parts exhibiting a variable degree of vulnerability in PD, searching for qualitative similarities and dissimilarities within PD affected regions (i.e. locus coeruleus), or unaffected monoaminergic neuron populations (i.e. VTA). Of note, TMT chemical tagging technology used in this project to obtain simultaneous qualitative and quantitative data from up to six samples in a single experiment is known to reduce protein identification rates. Shotgun proteomic analyses of unlabeled individual samples might enhance proteome coverage.

3. Identifying proteome alterations in the SN of PD patients

Altogether, our proteomic experiments revealed a set of proteins and pathways deregulated in PD, directly or indirectly involved in the neurodegenerative process as a cause or a consequence of it. The 2-DE comparative workflow (Chapter IV) resulted in the findings of 32 spots differentially expressed - 14 overexpressed and 18 underexpressed - in the PD (n=3) versus

175 the control (n=3) groups with a fold change cutoff over 1.4. Among them, seventeen spots were unambiguously identified. The sixplex TMT shotgun quantitative analysis (Chapter V) performed on a different set of patients - except for patient P2 which corresponds to 2-DE patient P3, yielded 204 differential protein groups with 96 over- and 108 underexpressed in PD (n=3) versus control (n=3) groups. When setting an arbitrary cutoff at a fold change higher than 1.3 to look at the most differential proteins, 11 proteins were up- and 10 proteins downregulated in PD.

As discussed in the next sections, our data confirmed the involvement of mitochondrial dysfunction, energy metabolism impairment, oxidative stress, cytoskeleton and vesicular defect, synaptic dysfunction, protein homeostasis deregulation or inflammation, previously found to be involved in PD pathogenesis by proteomics or other approaches such as transcriptomics. They also suggested some less conventional pathogenic pathways such as protein translation defects,

ER stress or abnormalities in the blood brain barrier (BBB) or extracellular matrix (ECM). A majority of the identified proteins were novel PD-related candidates including CNDP2, nebulette or seipin, verified by western blot and/or immunohistochemistry.

Mitochondrial dysfunction, energy metabolism impairment and oxidative stress

PD pathology has long been associated to mitochondrial dysfunction, impaired cellular energy metabolism and increased oxidative stress [14]. In sporadic PD their role is notably supported by the reduced mitochondrial activity and increased oxidative levels observed in PD brains [15-17]. The recent GWAS identification of variants in genes coding for mitochondrial proteins such as LRRK2, associated to a higher risk of developing PD [18]. Any failure in one of these three interconnected processes might be particularly critical for DA neuron demise and account for their specific vulnerability in PD. Indeed, DA neurons are known to rely on highest energy demand and more efficient antioxidant defense due to their particular morphological and physiological specificities [19, 20]. We reported many deregulated proteins potentially Chapter VI 177

linked to those pathogenic mechanisms in the SN of PD patients, with the most differentially expressed mapped on Figure 1. We found a decrease in multiple mitochondrial proteins including members of the respiratory chain complex in PD, pointing to a mitochondrial dysfunction and ATP depletion. Proteins linked to energy metabolism were found perturbed as well. Several enzymes involved in glycolysis or TCA cycle were underexpressed suggestive of energy depletion in PD, whereas others were found overexpressed which may indicate compensatory mechanisms to counteract defects in energy metabolism pathways (i.e glycolysis, mitochondrial oxidative phosphorylation) and preserve cellular ATP. We identified a prominent increase in proteins implicated in cell protection against oxidative free radical damage suggesting an increased oxidative insult in the SN of PD patients, possibly induced by mitochondrial dysfunction or energy metabolism failure. Some of the proteins falling in those categories were also found to be differentially expressed in other proteomic studies focusing on

SN, including ATP5H, Ferritin-L, GSTP-1 or peroxiredoxin-1, all increased in PD [11, 12, 21].

In addition, we identified a novel potential candidate, CNDP2, whose overexpression could be linked to oxidative stress, through its capacity to cleave antioxidant proteins such as carnosine or

GGH.

177

Figure 1. Potential links between mitochondrial dysfunction, energy metabolism failure and oxidative stress in the SN of PD patients. Our results point to a decreased mitochondrial function and energy metabolism as well as an increased oxidative insult in PD. These three processes are interconnected. Mitochondrial dysfunction leads to energy depletion and oxidative stress by ROS production. In turn, protein oxidation induces defects in mitochondrial or energy metabolic functions. Moreover, mechanisms to counteract oxidative stress and ATP depletion might be at work as well.

Synaptic, vesicular and cytoskeleton defects

Importantly, we were able to identify defects in the expression of several proteins involved in activities important for synaptic maintenance. A general trend towards a decrease in neuron-specific proteins such as syntaxin binding protein 1 (STXB1) or the post-synaptic density protein disk-larg homolog 2 (DLG2) for instance, was generally observed by us as well as others

[12, 22]. We also observed a decrease in soluble N-ethylmaleimide-sensitive factor attachment

(SNAP) protein gamma (SNAPG), a member of the SNARE complex particularly important for neurotransmission through the mediation of vesicle fusion through exocytosis. Vesicular defects might also occur elsewhere in the cell leading to protein homeostasis disruption and protein aggregation, ultimately perturbing neuronal function. A new link between PD and protein sorting was recently established with the discovery of mutations in the retromer complex VPS35 protein in late onset PD families [23]. Interestingly, we identified for the first time the overexpression of Chapter VI 179

vacuolar protein sorting 29 (VPS29), another member of this complex which is central for endosomal-lysosomal trafficking and membrane associated proteins recycling.

Cytoskeletal proteins, important for maintaining neuronal shape, vesicles/organelles trafficking or synaptic signaling, were already demonstrated to be involved in PD [24-26] and important components of LB [27]. Cytoskeleton alterations could disrupt axonal transport of various cellular proteins and organelles such as mitochondria leading to energy depletion and synaptic impairment, which may severely compromise the function and survival of high-energy demanding DA neurons. We found a preferential increase in cytoskeletal components including nebulette (NEBL) as well as moesin or coactosin-like protein also identified in other proteomic analyses [11, 12, 21] and a decrease in other proteins such as tubulin-8 (TUBA8) or neuron- enriched tubulin binding dihydropyrimidinase-like protein 2 (DPYSL2). Altogether, our results are consistent with a defective synaptic function in the SN of PD, potentially induced by alterations in selected proteins associated to cytoskeleton and vesicular transport. Post-synaptic abnormalities (i.e DLG2) may indicate rearrangements of afferent synapses in the SN.

Protein homeostasis and aggregation

Intracellular accumulation of misfolded or abnormal proteins such α-SYN is central to neurodegenerative diseases and results in the formation of LB in PD. Our data point to defects in several mechanisms known to promote protein aggregation, including protein processing

(expression level, PTMs), synthesis, transport or ER stress. The observation that increased levels of wild-type pre-synaptic α-SYN protein were sufficient to induce neurodegeneration in some genetic forms of PD [28-32] provided the rationale for searching for other aggregation-prone proteins, whose overexpression may participate in inclusion formation in sporadic PD. We identified more than a hundred proteins overexpressed in the SN of PD, some of which might accumulate pathogenically. To better delineate the potential aggregation-prone candidates, we compared our protein dataset with the extensive list of proteins previously identified in LBs

179

(Table 1) [33, 34]. Overall we identified 37 out of the 110 LB components reported in

Wakabayashi et al. and 99 of the 156 proteins reported in the proteomic analysis of cortical LBs by Leverenz et al. [33, 34]. We found some of those LB-associated proteins to be overexpressed in PD such as the cytoskeletal protein vimentin (Table 1), which may participate in the aggregation process. We did not identify overexpression of the classical LB components ubiquitin or α-SYN, similarly to other proteomic studies [10-12, 22]. This may be explained by several hypotheses. First, it is possible that α-SYN is not as central in sporadic PD as it is in familial forms of PD characterized by α-SYN gene mutation or multiplication. Second, because the number of DA neuronal cells is dramatically reduced in PD, the overexpression of synaptic α-

SYN in surviving DA neuronal PD cells may be compensated by the higher number of healthy neuronal cells in control patients. This hypothesis might as well account for the reduced expression observed in neuronal proteins previously associated to LB (Table 1) or not, such as the aggregation prone protein seipin (BSCL2). Finally, it is also possible that the methodology that we used did not allow a proper solubilization of LBs or protein aggregates, and that the observed upregulation was unrelated to them.

Table 1. Differential proteins identified either by our TMT or 2-DE proteomic workflows, that were also reported to be in LBs [33, 34]. Abbreviations: + = present, - = absent, NS = non significant; ID = identified

SN differential Protein name Gene name Brainstem LBs Cortical LBs TMT/2DE αB-Crystallin CRYAB − +/− ↑ / NS 14-3-3 protein (eta) YWHAH + + ↑ / NS Basic fibroblast growth factor FGF1 +/− − ↑ / no ID Creatine kinase B-type CKB − + NS/ ↑ Guanine nucleotide-binding protein G(I)/G(S)/G(T) sub beta-1 GNB1 − + NS/ ↑ Heat-shock proteins (i.e. 70) HSPA1A + + ↑ / NS Superoxide dismutase 2 SOD2 +/− ND NS/ ↑ Vimentin VIM − + ↑ / no ID Elongation factor 1-alpha 2 EEF1A2 − + ↓ / no ID Synaptophysin SYP +/− +/− ↓ / no ID Syntaxin-binding protein 1 STXBP1 − + NS/ ↓ Synapsin-1 SYN1 − + ↓ / no ID Tubulin TUBA8 + ND ↓ / NS Chapter VI 181

We also identified defects in protein synthesis and folding, with the reduction of translation elongation factor 1 alpha 2 (EF1A2) or endoplasmic reticulum protein 29 (ERP29) in

PD, which could account for the decreased expression levels in several proteins including ER- resident protein seipin (BSCL2). Misfolded seipin was shown to accumulate in the ER and trigger

ER stress, which in turn induces a diminution in translation, together with the unfolded protein response (UPR) to enhance protein degradation and finally apoptosis [35].

We did not directly observe impairments in protein degradation, a process known to be impaired in PD [36, 37]. However, the identified defects in energy metabolism failure and mitochondrial function may indirectly impact ATP-dependent UPS function. Overexpression of

VPS29 may indicate an increased transport of lysosomal hydrolases and potentially enhanced

ALP involved in the degradation of aggregates. Interestingly, we found the overexpression of ubiquitin-conjugating enzyme E2N (UBE2N), which could represent a protective attempt to clear inclusions by ALP through K63 polyub attachment [38].

Non-neuronal protein deregulation and proteins of unknown function

Abnormalities in neurons’ surroundings, including glial cell populations, BBB or ECM can critically influence positively or negatively the fate of resident neuronal cells. Evidence suggests that chronic inflammation processes mainly mediated by activated microglia can initiate or amplify neurodegeneration in PD, through the release of toxic factors (i.e. pro-inflammatory factors, ROS, NO) with some protection offered by the use of NSAIDS in patients [5, 39].

Accordingly, we as well as others, identified the overexpression of glial fibrillary acidic protein

(GFAP) reflecting the mild gliosis occurring in PD [11]. We found a pronounced deregulation in proteins potentially involved in the microglia clearance of aggregated proteins and apoptotic neurons including signal regulatory protein β-1 (SIRPB1) and annexin A1 (ANXA1) being either protective or pathogenic. Peripheral immune response, which can trigger inflammation, has been implicated in PD pathogenesis as well. For instance, IgG but not IgM were recently shown

181 to strongly immunolabel LBs [40]. The decrease in immunoglobulin mu chain C (IgM) that we observed in the SN of PD patients could be indicative of an attempt to reduce the inflammation process.

A few studies suggested that changes in the BBB occur in PD patients [41]. We found an abnormality in tight junction protein ZO2 expression, which could modulate communication between the CNS and peripheral immune systems, allowing endogenous antibodies and inflammatory molecules to cross the BBB and target DA neurons. Alterations of the ECM matrix may affect synaptic morphology and function [42]. We found a deregulation in ECM component neurocan, and nebulette involved in focal adhesions linking actin cytoskeletal network with

ECM. Our results highlighted the relevance of two less conventional hypotheses involving BBB and ECM in PD pathogenesis.

We identified several other differentially expressed proteins in the SN of PD patients, whose function and potential link with PD have not been determined yet. These include ornithine aminotransferase (OAT) or 14 kDa phosphohistidine phosphatase (PHPT1) found by our two proteomic approaches, as well as gamma glutamyl hydrolase (GGH). Unraveling the biological role of these proteins could lead to the exploration of novel hypotheses concerning PD pathogenesis. A summary of the highest protein changes observed in PD, classified according to the potential pathogenic mechanisms associated with them is presented in Figure 2.

Chapter VI 183

Figure 2. Potential pathogenic pathways involved in PD. This scheme presents the potential pathogenic involvement of the most differential proteins found in the SN of PD patients, in the context of their biological function. Proteins either overexpressed (red) or underexpressed (blue) are represented by their gene names. Four proteins highlighted in bold were found differential in both 2-DE (italic) and shotgun approaches.

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4. Interpreting proteomic results: some issues

4.1. Verification of candidates of interest

Proteomics generates large lists of proteins with concomitant quantitative data that need to be further verified using independent and reliable methods such as WB and IHC. We evaluated the differential protein expression levels of selected candidates, whose link with PD was either to be established (i.e. CNDP2, seipin, nebulette and GGH) or already assessed with controversial expression levels reported in the literature (i.e. FTL). Overall, our WB and IHC verification experiments confirmed our proteomic findings. For a more convincing assessment of nebulette and GGH expression levels, for which none of the tested antibodies worked in WB, IHC analysis (n=2) would need to be performed in a larger series of patients.

Interestingly, IHC analysis indicated a preferential localization of CNDP2, nebulette and

GGH inside neurons, confirming the ability of our approach to detect neuron-specific changes.

Importantly, similar levels of seipin and ferritin were found in the incidental (n=1) and classical

(n=3) PD cases, suggesting that the incidental subject represents a true pre-clinical form of PD.

This observation has two main crucial implications for our project. First, it constitutes a valuable proof that some of the observed changes in the SN are unrelated to PD treatment, as the incidental case was not under L-dopa or any other PD medications. Second, it provides the rationale for studying late PD stage brains, which may reflect as well changes occurring early in

PD pathogenesis, a particularly important finding in the context of early PD biomarker research.

4.2. The nature of the changes

Using a global whole tissue approach, signals coming from mixed cell populations making up SN tissue were analyzed together. As a major drawback, it was almost impossible to assess the contribution of each nigral subpopulation such as DA neuronal cells (approximately 10% in controls) and major glial cells (90%) to the total signal, except for proteins recognized to be confined to a particular cell type. As a consequence, changes in proteins expressed by multiple Chapter VI 185

cell types but occurring in specific nigral subpopulations (i.e. DA neurons) were probably attenuated or totally masked, translating into smaller fold change or even no significant ratios.

For example, signals from DA neuronal cell population might be diluted 10 times and become hardly detectable. Complementary IHC analyses have helped determining the nature of some changes, with CNDP2, nebulette or GGH expression found to be mainly neuronal.

Moreover, with a tissue-based normalization (i.e tissue weight and total protein concentration), the total number of DA neurons present in PD samples was theoretically reduced of 70% compared to controls. Thus, the overexpression of proteins in surviving PD DA neurons may be counteracted by the higher number of cells containing the given protein in controls. For example, CNDP2 and nebulette proteins found to be predominantly expressed in neurons by IHC, might for example exhibit a real much higher increase. This can explain as well why the neuronal protein α-SYN, known to be overexpressed in PD, did not exhibit a differential expression change.

When interpreting the hereby differential data, we have tried to establish links between proteins in the context of their biological function and some possible pathogenic processes associated to PD. However, some of these changes could be unrelated to the cause of PD pathogenesis but rather a consequence of the neurodegenerative process. GGH for instance, observed to be decreased in PD and shown to localize in DA neurons by IHC, did not seem to exhibit a particular decrease at the individual cell level, but rather in the total number of cells.

Thus, the observed underexpression in several neuronal proteins might reflect neuronal loss occurring in PD.

When looking overall at the list of differential proteins hereby presented, a general trend is observed for low significance cutoffs (around 1.4 for 2-DE and 1.1 for TMT) and relatively small fold changes, with only very few being over 2. This is indicative of a small intra- and inter-group variability, pointing to great proteome homogeneity irrespective of sample group. Such little proteomic differences between PD and control groups could be attributed to

185 the factors above mentioned such as DA neuron signal dilution and are relevant with an adequate normalization. Moreover, we might have preferentially detected changes coming from the highest abundant proteins, not necessarily those exhibiting the greatest levels of protein expression change. Signals from low abundant proteins, either glial or neuronal, might stand beyond the dynamic range and sensitivity limits of our proteomic approaches. For example, we did not find any significant alteration of the classical DA neuron protein

(TH), identified in the TMT workflow only. However, TH was effectively reduced in PD samples and the “biological p-value” reflecting inter-group variability was significant. The “ratio p-value” was however not significant, indicating that signals were not sufficient to ensure a good ratio accuracy, which could be due to weak signals coming from less abundant neuronal proteins.

4.3. Concordance within proteomic studies and other “omics”

Comparative proteomic studies have often received little attention from the neurosciences community due to several reasons including the absence of well-defined hypotheses and the low concordance rates observed between them. It is generally difficult to compare proteomic studies together, as many sources of variability can drastically influence the final outcome. First, samples themselves are greatly heterogeneous, with divergences inherent to patient’s history, co-morbidities, PD subtype or disease duration, all hardly controllable parameters. Tissue quality can also affect protein changes, when PMD delays are too important or different between groups. Second, the lack of standardized protocols for sample preparation

(i.e. dissection, solubilization buffers) and analysis may prevent inter-laboratory comparisons as well. In fact, the plethora of existing analytical methods may lead to variability in the identified proteome. This translates into small overlaps in protein identifications generally observed across proteomic studies. For example, more than 1200 proteins of our total dataset were not identified in the few other proteomic investigations studying SN [10-12]. Third, the generally small number of samples examined by proteomic is associated to a low statistical power for Chapter VI 187

detecting differentially expressed proteins. The lack of stringent criteria to select differential proteins together with the absence of further result validation, an almost impossible task given the usually large differential protein datasets, may lead to the identification of false positives and false negative candidates.

Consequently, only a small percentage of proteins were found similarly differential across the few proteomic studies analyzing SN, although a high proportion of non-differential proteins were concordant between them. Four proteins (i.e. CNDP2, PHPT1, OAT, FTL) were identified in both our 2-DE and TMT workflows, exhibiting changes in the same direction. Twenty other proteins identified in one of our two workflows were found similarly differential by others

[11, 12]. While the majority exhibited concordant ratios (i.e. ATP5H, Neurocan, GFAP) a few were discordant (i.e. IgM, TUBA8). These differential proteins found inversely expressed across studies may indicate the presence of different protein isoforms differentially regulated that may still participate in the same pathogenic mechanism. Indeed, PD is known to be a heterogeneous disease and distinct alterations in common pathways may induce a common phenotype. For example, different mutations and multiplications of α-SYN all result in familial PD [43, 44].

Interestingly, we could identify a number of similarities between our proteomic results and those obtained by transcriptomics validating somehow our approach, with some examples given in Table 2. We identified 80 of the 227 genes found differentially expressed in the SN of PD patients by Bossers et al. [45]. Of them, 11 were also differential, all with concordant ratios. We identified 199 of the 1044 genes exhibiting a differential expression in Simunovic et al. study, which profiled specifically the transcriptome of LCM isolated DA neurons in PD versus controls

[46]. We found 27 of them similarly differential, with this time 15 concordant ratios but 13 non- concordant ones indicating changes unrelated to neurons. It is also possible that mRNA levels do not correlate with protein levels for those proteins [47]. Three proteins including LRPPRC were found in common between proteomics and those selected transcriptomics studies, with concordant ratios. Moreover, common pathways were found deregulated across both proteomic

187 and transcritpomic studies including neurotransmission, synaptic dynamics or mitochondrial function. Similarly to what is generally thought for transcriptomics data, the absence of concordance between proteomic studies could be due to the utilization of protein list for comparisons, rather than standardized pathways, which could indicate the involvement of common pathogenic mechanisms. To conclude, instead of being taken as conflictual, the results obtained in proteomics may rather be seen globally, each proteomic study identifying a fraction of the changes occurring in the SN.

Table 2. Comparison between proteomics and transcriptomics results in PD. Examples of proteins found differentially expressed by our proteomic approaches, whose mRNA levels were similarly found differential by transcriptomics in the SN [45] or DA neurons [46] of PD patients versus controls. Proteomics Transcriptomics Protein Gene SN SN LCM DA neuron Protein PD/C RNA PD/C RNA PD/C Transmembrane protein 35 TMEM35 0.7 0.4 / Vimentin VIM 1.2 2.2 / Leucine-rich PPR-motif containing LRPPRC 0.8 0.6 0.5 synapsin I SYN1 0.9 / 0.6 ornithine aminotransferase OAT 0.9 / 0.6

5. Beyond SN: a common pathogenic mechanism underlying PD?

Although neurodegeneration appears to be more acute in the SN, PD is now recognized to be a much wider multisystem disorder affecting many CNS regions. According to Braak, PD pathology may progress rostro-caudally to reach the locus coeruleus (LoC) before affecting the

SN as well as higher brainstem and cortical regions, possibly in a prion-like manner [48, 49].

Interestingly, some of our differential proteins including mitochondrial LRPPRC, LB-associated vimentin or seipin were found similarly differential in a proteomic study analyzing the LoC [22], strengthening their role in PD. Indeed, common pathological mechanisms including mitochondrial dysfunction, cytoskeletal defect or ER stress, may occur in different PD- Chapter VI 189

susceptible brain regions resulting in protein aggregation and neurodegeneration. Of note, two of these proteins, LRPPRC and seipin were already linked to other neurological conditions, indicating possible connections between the pathological mechanisms of these diseases and PD.

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PERSPECTIVES

1. Digging deeper in the SN proteome

Post-mortem versus in vivo SN samples

Human brain tissue analyses are essential to uncover PD-specific abnormalities in the absence of any fully satisfying animal model. Issues regarding the availability, number and quality of autopsy tissue samples can now be partially overcome through facilitated access to existing brain banks - such as the Queen Square Brain Bank for Neurological Disorders (QSBB) - which ensure the collection of well characterized and preserved brain tissues (PMD<12h). The setting up of a brain bank in Geneva or in a larger scale Switzerland was already discussed and might be created in the years to come, when logistics and financial aspects will be assessed.

Recently, a novel technology was developed which might allow the molecular imprint of nigral tissues from living PD patients. Taking advantage of the temporary access to cerebral regions during the implantation of DBS electrodes for PD treatment, the approach allows the capture of small tissue amounts (i.e.20 µg proteins) using a chemically modified micro silicon chip placed at the bottom of the surgical stylet [50]. The tool already permitted the exploration of human STN proteome, with more than 1200 protein identified (Zaccaria A. et al, submitted manuscript). The technology might be transferred into Geneva University Hospitals by the end of 2013. The use of in vivo nigral imprinting would reduce PMD to a few minutes avoiding protein degradation and may allow the observation of changes occurring early in PD course, although control samples might be more difficult to obtain for comparisons.

Isolating nigral sub-proteomes: the DA neuron

The great complexity and cellular heterogeneity characterizing human brain regions may be further addressed by additional cellular and subcellular fractionation steps. In the SN, the Chapter VI 191

mixed cell populations together with the characteristic neuronal DA loss in PD may have obscured the identification and quantification of subtle changes limited to DA neurons. Laser- capture microdissection (LCM) offers the possibility to specifically isolate and investigate separately small defined areas including neurons, facilitating data interpretation. The use of LCM in proteomics has long been hampered by the necessity to collect large number of cells

(between 10’000 and 100’000) to obtain sufficient amounts of proteins for analysis and the lack of protein amplification systems. The emergence of more sensitive MS techniques (i.e. LTQ-

Orbitrap) and automated methods to collect cells have in part overcome these limitations, recently allowing the analysis of the cortical LB proteome in patients with dementia with LB [34].

The next step of this project will be to establish and compare the proteomes of LCM-isolated DA neurons (i.e approximately 2500 cells/patient) in PD versus control patients by unbiased shotgun proteomic approaches involving label-free quantification. Besides allowing the distinction between neuronal and glial protein expression changes, targeting the specific cell populations selectively affected by PD pathology may provide considerable insights into PD pathogenesis.

Interestingly, we may also try to compare the proteome of PD LB-bearing and LB-free DA neurons to control neurons to go even deeper into the specific pathophysiological features of DA neurons at the basis of their susceptibility to PD.

The enrichment in various subcellular fractions applied at a tissular or at a cellular level could increase the coverage of proteomes of interest. In particular, the proteomic analysis of mitochondria-enriched or synaptosomal-enriched fractions in PD patients versus controls could inform us on the pathological molecular mechanisms occurring specifically in those cellular compartments particularly relevant for PD pathogenesis. In addition, it might be interesting to examine protein post-translational modifications such as oxidations, phosphorylation or ubiquitination, known to play important roles in PD pathology and in aggregation process in particular.

191

Integromics

The recent emergence of a variety of “omics” strategies including proteomics but also transcriptomics, metabolomics or lipidomics has boost the exploration of the molecular mechanisms at the basis of PD. In particular, transcriptomics analyses of nigral tissues and DA neurons were already proved successful in the context of PD pathogenesis [45, 46]. We thus project to obtain the transcriptomic profiles of the future LCM-dissected DA neurons in PD versus control patients to complement the proteomic data and increase the strength of our study through the eventual involvement of common pathways found in both approaches. The bioinformatics integration of both transcriptomics and proteomics datasets through system biology approaches might provide a more comprehensive picture of the specific pathological mechanisms occurring in the highly selective DA neuron population.

2. Functional biology

In this thesis, we reported the differential expression of several novel potentially pathogenic candidates in the SN of PD patients. Their biological evaluation is critical to gain insights into the molecular mechanisms underlying PD pathology through a better knowledge of their physiological functions and dysfunction in PD. We found that CNDP2 dipeptidase was overexpressed mainly in surviving DA neurons of PD patients. These observations suggested for the first time the involvement of CNDP2 in PD, possibly through mechanisms related to oxidative stress as CNDP2 is known to cleave several anti-oxidants.

Cellular studies using a PD model may provide a simple, quick and robust method to dissect the functions of CNDP2 in PD pathogenesis. The human SH-SY5Y neuroblastoma cell line is one of the most popular cell model used in PD research as it possess many characteristics of DA neurons and can be differentiated into mature DA neurons. Exposed to low concentrations of rotenone, a neurotoxin inhibiting mitochondrial complex I, this model was shown to reproduce

PD pathological features, affecting neuronal survival, mitochondrial function and protein Chapter VI 193

aggregation [51]. We would like to extend our proteomic findings to such model, to see whether

CNDP2 is overexpressed with increased rotenone concentrations. To gain more insights into

CNDP2 role in PD, we may manipulate in vitro its expression levels using siRNA for silencing or transgenes for over-expression and evaluate the effects on rotenone-mediated neurotoxicity.

Observations that CNDP2 under- or over-expression respectively attenuate and enhance rotenone-mediated neurotoxicity measured by various means (i.e. cell viability, protein oxidation or aggregation), could argue for a causative toxic rather than a protective role of

CNDP2 in PD pathogenesis.

3. Translating basic sciences into clinical applications

Without any curative treatment, PD represents an increasing challenge for healthcare systems in the context of the general population aging. Effective therapeutic interventions directly impacting the disease biology are urgently needed, that will slow or ideally stop the inexorable progression of the neurodegenerative process. Advances in the understanding of the specific pathological actors mediating molecular events at the basis of neurodegeneration in PD such as those provided by proteomics may uncover new avenues for treatment and perhaps prevention of the disease.

At present, efforts in translational medicine applied to PD have mainly focused on turning α-SYN knowledge into drugs, given the considerable amounts of evidence acquired these past 15 years for the causative role of this protein in PD. Some of the possible therapeutic strategies imagined to block α-SYN pathological effects are summarized in Figure 3. For example,

α-SYN removal has the potential to modify PD progression. Experimental data have suggested that immunization approach promotes α-SYN clearance through lysosomal pathway and reduce neurodegeneration in animal models [52]. Promisingly, the first PD vaccine named PD01 has been developed by AFFITOPE and has just entered clinical phase I in 2012[53]. In the future, some of the molecules highlighted in this project might prove to be interesting therapeutic

193 targets (i.e. CNDP2), although their involvement in the disease pathogenesis still need to be determined by molecular biology experiments. Alternatively, this work might suggest new therapeutic avenues involving ER stress or ECM and BBB defects.

Figure 3. Possible therapeutic interventions in PD. Stefanis et al. Cold Spring Harb Perspect Med,

2012

Finally, reliable biomarkers allowing a definitive diagnosis early in the disease course, a cornerstone for the establishment of future disease-modifying treatments, are still critically needed as well. Pathological abnormalities in the brain are expected to be reflected in CSF proteome, due to its particular connective role with brain. α-SYN oligomers levels have been measured in CSF but conflicting results may preclude their use as PD biomarkers [54]. Brain tissue proteomic discovery screening could provide a valuable source of neuropathologically- derived biomarkers. Given the possible pathological role of CNDP2 in PD, we tentatively assessed its potential as a marker of PD by WB in the CSF of PD (n=7) versus healthy (n=7) patients. No significant differences were observed between groups, potentially due to a late- Chapter VI 195

onset phenotype of CNDP2 overexpression, the dilution of CNDP2 signal thus hardly detectable or an insufficient number of patients tested. It is expected that some other pathogenic candidates might be better markers of PD and should be further tested by WB or ELISA. For example, Seipin or Ferritin-L whose alterations might already appear in the pre-clinical phase, might serve as early PD biomarkers. Their relative specificity and ability to discriminate true PD cases should be tested with control groups exhibiting other neurological conditions such as seipinopathy or showing clinical overlap with PD (ie. MSA, PSP, CBD etc.).

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CONCLUSION

During the last decades, multiple cellular and molecular mechanisms were proposed to account for nigral pathology, responsible for the main PD motor symptoms. However, the precise etiopathology underlying the neurodegenerative process remains enigmatic, precluding the development of curative interventions. To gain insights into PD pathogenesis we analyzed SN autopsy tissues by proteomics, allowing the unbiased global analysis of nigral proteomes from

PD patients compared to controls.

The project resulted in a great amount of data particularly relevant in the context of basic but also translational PD research. First, we obtained the more comprehensive picture of the human SN proteome, which constitutes a first step towards a better understanding of the SN function and its specific vulnerability in PD. Second, we successfully confirmed the involvement of existing theories on PD pathogenesis including mitochondrial dysfunction, oxidative stress, cytoskeleton defect, synaptic dysfunction, protein homeostasis deregulation or inflammation.

Third, we suggested some less conventional pathogenic pathways such as protein translation defects, endoplasmic reticulum stress as well as abnormalities in the blood brain barrier or extracellular matrix. Fourth, our approach highlighted overall a majority of novel potential candidates engaged in PD pathological process, either as a cause or a consequence of it, including CNDP2, seipin or nebulette. To conclude, the complex proteome alterations observed in the SN of PD patients provide further insights into the pivotal pathogenic processes engaged in PD and may ultimately lead to the development of new tools for the treatment and diagnosis of PD.

Chapter VI 197

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