Дисертації з теми "Α-synuclein aggregation"

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
It is widely known that α-synuclein (aSyn) is an amyloidogenic protein prone to aggregation. This protein is found in specific inclusions named Lewy bodies in the surviving neurons of Parkinsons’s disease patients and other synucleinopathy brains. This aggregation process is greatly affected by different post-translational modifications, such as phosphorylation, acetylation, and glycation. Lately it was shown that aSyn oligomeric species are more toxic than the inclusion bodies. Heat shock proteins (HSPs) are molecular chaperones able to modulate the folding and refolding of proteins. Its overexpression in Parkinson’s disease models reduces and prevents aSyn aggregation. As the reduction of aSyn aggregation can lead to an eventual accumulation of oligomeric species which may cause cell damage, the main goal of this work is to better understand the role of HSPs in aSyn oligomer formation, clarifying which are the aSyn resulting species formed in the presence of HSPs. Moreover, as glycation is suggested to accelerate abnormal protein deposition, we aimed to investigate how HSPs interfere with the oligomerization process of glycated aSyn. In this study Hsp70 seemed to induce recombinant aSyn oligomerization, generating higher molecular weight species with no associated toxicity. On the other hand, Hsp27 reduced aSyn oligomerization in vitro possibly by inducing the formation of non-reactive small oligomers. MGO glycation increased protein aggregation and cell death. Interestingly, Hsp27 overexpression reversed glycated aSyn aggregation and its associated toxicity. These results demonstrate the importance of HSPs modulation as a possible target of Parkinson’s disease therapeutics.

Les maladies neurodégénératives, telles que les maladies d'Alzheimer, de Parkinson et de Huntington, sont des maladies incurables caractérisées par une perte progressive de neurones, entraînant des déficits cognitifs et moteurs chez les patients. Le développement de traitements efficaces est actuellement limité par notre compréhension incomplète des mécanismes à l’origine de l’initiation et de la progression de la maladie. Une caractéristique dominante observée dans les tissus affectés par les ces maladies est la présence de protéines agrégées, qui sont devenues la marque de maladies telles que la maladie de Parkinson et la maladie d’Alzheimer. Ainsi, le processus d’agrégation jouerait un rôle central dans la pathogenèse de la maladie. Cependant, les mécanismes spécifiques qui sous-tendent la transition des protéines solubles vers leur état agrégé restent insaisissables. Des recherches récentes ont proposé la séparation de phase (PS) comme étape intermédiaire critique dans la formation d'agrégats de protéines. La PS est un phénomène physique dans lequel certaines protéines subissent une transition de phase, formant des gouttelettes distinctes ayant des propriétés de liquide dans l'environnement cellulaire. Les condensats biomoléculaires sont impliqués dans divers processus physiologiques, mais sont de plus en plus supposes d’être sujets à des comportements aberrants pouvant déclencher des conditions pathologiques. Bien que prometteuse, l’étude du rôle du PS dans le contexte des maladies neurodégénératives reste une tâche difficile en raison de la composition complexe des condensats cellulaires. Ils sont constitués de dizaines, voire de centaines de biomolécules différentes, notamment des protéines, des acides nucléiques et des lipides, créant un milieu complexe qui exige des approches de recherche innovantes. Pour améliorer l'étude de la PS dans le contexte des maladies neurodégénératives, nous avons développé une méthode de formation et de dissolution contrôlées de condensats biomoléculaires enrichis en protéines impliquées dans l'agrégation pathologique. Tout d’abord, nous avons créé des condensats contenant de l’α-synucléine (α-Syn), le principal facteur pathologique de la maladie de Parkinson et d’autres pathologies. L'α-Syn est connue comme une protéine de type prion, ce qui signifie que son agrégat se propage de cellule en cellule et modèle l'agrégation de l'α-Syn cytosolique soluble, favorisant ainsi la progression de la maladie. Cependant, on sait peu de choses sur ce phénomène en ce qui concerne l’état condensé de la protéine. Pour simuler cela, nous avons exposé des cellules exprimant nos condensats α-Syn à des agrégats α-Syn préformés sous forme fibrillaire. Nous avons observé que les fibrilles déclenchaient la transition des condensats de leur état liquide à une forme agrégée ayant des propriétés solides et présentant des marqueurs biochimiques caractéristiques des amyloïdes. Cela nous a permis de proposer un modèle dans lequel la phase condensée de α-Syn accélère la propagation des agrégats pathologiques, en fournissant un pool de protéines concentrées pouvant subir plus facilement une transition amyloïde via le mécanisme de type prion. Par la suite, nous avons construit des condensats artificiels enrichis en α-Syn et la synapsine. Dans les neurones, α-Syn et la synapsine interagissent aux extrémités présynaptiques, où ils jouent un rôle important dans la libération des neurotransmetteurs en contrôlant le regroupement, le trafic et la libération de conteneurs de neurotransmetteurs appelés vésicules synaptiques. Nos condensats d'α-Syn/synapsine étaient eux également soumis à une transition liquide-solide médiée par les fibrilles d'α-Syn
Neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's disease, are incurable illnesses characterized by the progressive degeneration of neurons, leading to cognitive and motor deficits in affected individuals. The development of effective treatments is currently limited by our incomplete understanding of the underlying mechanisms that drive disease initiation and progression. One prevailing characteristic observed in the tissues affected by neurodegenerative diseases is the presence of aggregated proteins, which have become hallmarks of diseases such as Parkinson’s and Alzheimer’s. Thus, the aggregation process is believed to play a pivotal role in disease pathogenesis. However, the specific mechanisms underlying the transition of soluble proteins to their aggregated state remain elusive. Recent research has proposed phase separation (PS) as a critical intermediate step in the formation of protein aggregates. PS is a physical phenomenon wherein certain proteins undergo a phase transition, forming distinct liquid-like droplets within the cellular environment. Biomolecular condensates are involved in various physiological cellular processes, but are also increasingly believed to be subject to aberrant behaviours that can trigger pathological conditions. Although promising, studying the role of PS in the context of neurodegenerative diseases is a challenging task, due to the intricate composition of cellular condensates. They consist of tens to hundreds of different biomolecules, including proteins, nucleic acids, and lipids, creating a complex milieu that demands innovative research approaches. To improve the study of LLPS in the context of neurodegenerative diseases, we have developed a method for the controlled formation and dissolution of biomolecular condensates enriched in proteins involved in pathological aggregation. First, we created condensates containing α-synuclein (α-Syn), the main pathological factor in Parkinson’s disease and other pathologies. α-Syn is known as a prion-like protein, meaning that its aggregates spreads from cell to cell and template the aggregation of soluble cytosolic α-Syn, promoting the progression of the disease. However, little is known about this phenomenon with respect to the condensed state of the protein. To simulate this phenomenon, we exposed cells expressing our α-Syn condensates to preformed α-Syn aggregates in fibrillar form. We observed that fibrils triggered the transition of condensates from their liquid-like state to an aggregated form with solid-like properties and exhibiting biochemical markers characteristic of amyloids. This allowed us to propose a model where the condensed phase of α-Syn speeds up the propagation of pathological aggregates, by providing a pool of concentrated protein that can undergo more easily an amyloid transition via the prion-like pathway. Subsequently, we have built artificial condensates enriched in α-Syn together with synapsin. In neurons, α-Syn and synapsin interact at the presynaptic termini, where they play an important role in the release of neurotransmitters by controlling the clustering, trafficking and release of membrane-enclosed neurotransmitter containers called synaptic vesicles. In our setting α-Syn/synapsin condensates were also subject to a liquid-to-solid transition mediated by α-Syn fibrils

Neurodegeneration in Dementia with Lewy Bodies (DLB) and Parkinson’s disease (PD) is associated with the formation of neuronal inclusion bodies composed mainly of aggregated α-synuclein (α-syn) protein. Aggregation may be associated with disturbed Ca2+ homeostasis and oxidative stress. Post-mortem studies have shown relative sparing of neurons in PD that are positive for the Ca2+ buffering protein, Calbindin-D28k (CB). CB has been shown to be induced by the hormonal form of vitamin D, Calcitriol, and could be induced by other vitamin D analogue such as Calcipotriol (Cp). Furthermore, recent cell culture and in vitro studies have shown that α-syn aggregation can be induced by potassium depolarization, hence we hypothesized that Cp may suppress their formation. We investigated the interplay between α-syn aggregation, expression of the calbindin-D28k (CB) Ca2+-buffering protein and oxidative stress in neurons by comparing DLB and “healthy” human brain tissue and examining a unilateral oxidative stress lesion model of -syn disease (rotenone mouse), using the combination of immunofluorescence double labelling and Western blot (WB) analysis. DLB cases showed a greater proportion of CB-positive (CB+) neurons in affected brain regions compared to normal cases. Lewy bodies were present predominantly in CB-negative (CB-) neurons and were virtually undetected in CB+ neurons.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
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Aggregation of α-synuclein (α-syn) into toxic fibrils is a pathogenic hallmark of Parkinson’s disease (PD). This research aimed to develop peptides capable of inhibiting α-syn aggregation using a semi-rational design combined with a multiplexed intracellular Protein-fragment Complementation Assay (PCA) library screening system. Successfully selected peptides must bind to full length α-syn and lower its toxicity to confer bacterial growth. PCA selected peptides were characterized using several biophysical assays and a cell viability assay. The peptides were screened using library templates based on α-syn71-82 initially and later on the α-syn45-54 region in which many key mutations associated with early onset PD are found. In both cases we targeted the peptide libraries at the wild type protein or again by using mutated versions of α-syn. Results demonstrate that some of those selected peptides had the effect of delaying or even preventing the aggregation process, with others providing more subtle effects in reversing the fully formed amyloid fibrils. PCA peptides selected against 71-82 region of wild type α-syn showed a moderate level of efficacy whereas against mutants, it showed a low level of efficacy in inhibiting amyloid fibril formation. In the final part of the study, the peptide selected against 45-54 region of wild type α-syn was capable of preventing the aggregation and reducing the amyloid cytotoxicity at an equimolar ratio. We have thus demonstrated that the PCA strategy can be used as a generalised method for deriving peptide antagonists of α-syn aggregation, together with a new region; α-syn45-54 as an inhibitor target and produced a peptide inhibitor expected to provide a scaffold for future drug candidates to slow or even prevent the onset of PD.

Les signatures histopathologiques de principales maladies neurodégénératives - maladie d'Alzheimer et la maladie de Parkinson - sont les enchevêtrements neurofibrillaires formés par la protéine tau et les corps de Lewy, formés par l'α-synucleine agrégée. Les mécanismes précis du repliement et de l'agrégation de ces protéines, pour la protéine tau comme pour l'α-synucleine, ne sont pas totalement compris à ce jour. Ici, nous nous sommes intéressés à cette question en utilisant des modèles in vitro et in vivo. En étudiant l'agrégation tau in vitro, nous avons mis en évidence un nouvel auto- assemblage réversible de tau, qui dépend de la température et de la présence d’ions zinc, et qui est a priori différent de l'agrégation de tau en présence d'inducteurs d'agrégation tels que l'héparine. Ce processus pourrait néanmoins être impliqué dans les premières étapes de l'agrégation pathologique de tau. Dans une deuxième partie nous avons développé des modèles murin pour étudier les dysfonctionnement de l’α-synucleine. Nous avons montré que l’α-synucleine est directement impliquée dans le développement embryonnaire de régions spécifiques du système nerveux, et qu'elle a des propriétés modulatrices seulement sur les neurones dopaminergiques de la substantia nigra, qui sont touchés dans la maladie de Parkinson.Les résultats obtenus dans nos études de deux protéines qui subissent une agrégation pathogène et forment des inclusions intracellulaires ont contribué à la compréhension des processus moléculaires et cellulaires associés à la dégénérescence neuronale, ce qui fournira de nouvelles pistes pour développer de nouvelles stratégies de thérapies de maladies neurodégénératives
The histopathological hallmarks of the most common neurodegenerative diseases – Alzheimer’s disease and Parkinson’s disease are neurofibrillary tangles formed by tau protein and Lewy bodies inclusions formed by aggregated α-synuclein. The formation and accumulation of these proteins into inclusions cause functional disruptions of the cytoskeleton and leads to neuronal degeneration. The precise mechanisms of tau and synuclein misfolding and aggregation leading to those cellulare incluses, even though very studied, are not fully understood neither for tau protein nor for α-synuclein.Here we have addressed this question using both in vitro and in vivo models. Investigating tau aggregation in vitro, we have found a reversible self-assembly of tau, which depends on temperature and is induced by zinc ions, which is different from the tau aggregation in the presence of aggregation-inducers such as heparin. This process could be implicated in the first steps of tau pathological aggregation. In a second part, we have developed a mouse model for studying the α-synuclein dysfunction. We have shown that α- synuclein is directly involved in the embryonic development of the specific regions of the nervous system, and that it has modulating effect only on the populations of dopaminergic neurons of substantia nigra, which are affected in Parkinson’s disease.Results obtained in our studies of two proteins that undergo pathogenic aggregation and form intracellular inclusions contributed to understanding of molecular and cellular processes associated with neuronal degeneration, which is important for the development of new disease-modifying therapies of neurodegenerative disorders

Protein conformational diseases (PCD) include a range of degenerative disorders in which specific peptides or proteins misfold and aberrantly self-assemble, eventually forming amyloid-like fibrils, which constitute the hallmark of many neurodegenerative diseases. Plenty of works demonstrated that amyloid aggregation is strongly influenced by several extrinsic factors, such as high concentrations of macromolecules or the presence of proteases and chaperons. Molecular chaperones have been recognised as key players in the avoidance of amyloid fibril formation and, in particular, recent evidences demonstrate that low levels of chaperone heat-shock protein 70 kDa (Hsp70) are strictly related to the formation of intra-neuronal inclusions associated with Parkinson‘s (PD) and polyglutamine (polyQ) diseases. Human Hsp70 is composed of two major functional domains connected by a conserved interdomain linker: the 44-kDa N-terminal nucleotide-binding domain (NBD), with ATPase activity, followed by the 30-kDa substrate-binding domain (SBD) that contains a C-terminal lid subdomain (LS). Using a battery of Hsp70 variants, including full-length Hsp70 and SBD truncated variants, we have been able to discover an interaction between the LS of SBD and the interdomain linker, which we propose could play an important role in the allosteric communication between NBD and SBD. Therefore, we analyzed the anti-amyloidogenic activity of Hsp70, using two model proteins: alpha-synuclein (AS), whose deposition in the brain is associated with PD and the polyQ protein ataxin-3 (AT3), the causative agent of the Machado-Joseph disease (MCD). We demonstrated Hsp70 is able to interact and stabilize pre-fibrillar species formed during amyloid aggregation and that the binding mechanism of these species is different from that of the monomeric protein. Plenty of evidence supports the idea that protein aggregation observed in in vitro experimental conditions is different from that naturally occurring in in vivo systems. This is also related with the fact that the high concentration of macromolecules present in the intra- and extra-cellular compartments, a condition known as molecular crowding (MC), strongly affects protein folding and aggregation. Here, we successfully employed Escherichia coli as in vivo model for studying the aggregation mechanism of the polyQ protein ataxin-3 (AT3) in the presence of MC. In particular we investigated the relationship between the aggregation pathway and cytotoxicity and we were able to characterize the kinetic of formation of aggregated toxic and non-toxic species of AT3. Our future efforts will be aimed to investigate in vivo Hsp70 action by analyzing structural and physiological features of AT3 aggregated species formed in the intracellular environment of E. coli that co-express Hsp70 under different conditions.

The pre-synaptic protein α-Synuclein (αS) is often linked to the pathology of Parkinson’s disease (PD), an age-related neurodegenerative disorder. Lewy bodies, the cytopathological hallmarks of PD, are found to be rich in aggregates of misfolded αS protein. Metal dyshomeostasis has also been linked to PD due to the accumulation of iron in the substantia nigra pars compacta, and diminished copper levels reported in this same region. Metal dyshomeostasis in the brain coupled with oxidative stress can enhance the aggregation of αS. Recently, it was confirmed that mammalian αS is universally acetylated at the N-terminus, a common post-translational modification in humans. The consequences of this modification have been understudied, and it is believed to impart a functional role under physiological conditions with respect to membrane-interactions and protein folding. In an attempt to elucidate the pathological mechanism behind PD with respect to the structural dynamics of the protein, our investigations were focused on physiologically prevalent, N-terminally acetylated αS (NAcαS) and its interaction with the most prevalent redox-active metal ions in the brain (iron and copper) under both aerobic and/or anaerobic conditions. The structural features associated with metal-bound NAcαS differed depending on the iron oxidation states, where under aerobic conditions Feᴵᴵ stabilized an oligomer-locked, anti-parallel right-twisted β-sheet conformation that could potentially impart toxicity to neurons. In contrast, Feᴵᴵᴵ promoted a fibrillar structure rich in parallel β-sheets. N-terminal capping also altered the Cuᴵᴵ coordination sphere and had a dramatic effect on protein aggregation. Parallel studies on NAcαS variants with different site mutations near the putative copper binding sites (ex: H50Q and F4W) indicated that preferential binding shifts upon changes in the side chain residues. In depth analysis of the electron structure of Cuᴵᴵ-bound NAcαS using electron paramagnetic resonance spectroscopy (EPR) revealed a coordination sphere of N3O1 that includes the H50 residue in the wild-type protein that shifts to an O4 coordination sphere at the C-terminus upon Cuᴵᴵ binding to the disease-relevant H50Q variant. Immunoblotting analyses revealed that copper-induced redox chemistry promoted O2-activation and the subsequent formation of dityrosine crosslinks, a post-translational modification identified as a biomarker of PD. EPR-detection of tyrosyl radical formation in the presence of Cuᴵ-bound NAcαS further supported this radical coupling mechanism. Intermolecular crosslinks within the fibrillar core of NAcαS as well as intramolecular crosslinks within the C-terminal region underpin the role of metal-dioxygen chemistry in PD-related pathology. The unique structural features resulting from iron vs copper coordination to NAcαS inspired studies directed at the synergistic effect of each individual metal species as revealed by photo-initiated crosslinking of NAcαS. C-terminal intramolecular tyrosine interactions were mainly impacted by the presence of both metals, which each have binding sites around the same region. These findings emphasize that protein dynamics, metal binding site conformational changes, as well as aggregation pathways can deviate drastically upon N-terminal acetylation of αS and that protein-metal interactions may play a vital role in PD etiology.

Abnormal protein aggregation has been implicated in the pathogenesis of many neurological disorders. This study focuses on the protein alpha-synuclein (α-syn), a pre- synaptic protein that is involved in a number of diseases collectively termed alpha- synucleinopathies, which include Parkinson’s disease (PD) and Multiple System Atrophy (MSA). α-syn aggregation and microscopically-visible α-syn-positive intracellular inclusion bodies are common features of these diseases, occurring in multiple cell types and localisation throughout the central nervous system. Although gene mutations in a variety of molecular pathways have been identified in rare familial forms, the majority of α- synucleinopathy cases are sporadic in origin and have a late onset (>60 years) and therefore it is important to study age related changes in neurochemistry and how these changes may be responsible for the neurodegeneration associated with α-syn aggregation. With aging, tightly regulated cellular processes start to lose the capacity to maintain homeostasis. It is known that there is increased level of oxidative stress in aged compared to young brains, and that intracellular free calcium (Ca2+) is increased at both the resting level and upon neuronal activation. This research is focused on these two processes: firstly the increase of intracellular free Ca2+; and secondly, the increase in oxidative stress.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Rab GTPases, working as essential controllers of cellular membrane dynamics, recently associated in diverse pathological pathways of Parkinson's disease (PD). In particular, RAB39B pathogenic variants cause intellectual disability (ID) and X-linked early-onset PD with α-Synuclein (α-Syn) brain pathology. Notably, RAB39B gene is specific to the brain, highly expressed in neurons mainly involved in intracellular vesicles trafficking in the ER-Golgi-endosome secretory pathways but it was found to be work in synapse formation and maintenance. The overall objective of this work is finding new insights about its unknown role in PD pathogenesis and which mechanism could lead to neuronal weakness in autophagic deficit of toxic cellular aggregates and presynaptic dopaminergic (DA) alteration. In order to identify RAB39B effectors and downstream key molecular functions, its interactome was identified thanks to the implementation of APEX2 labelling system directly on living neuronal culture. Through this biochemical approach, we establish a novel link between RAB39B and the retromer complexes, with functional consequences on cargo sorting like for M6PR, ATG9 or ATG9. Studying its interactors, we focused on vesicular and membrane trafficking function during the formation of the nascent autophagosome. We hypothesized that RAB39B loss-of-function could lead to a defect in autophagic degradation of α-Syn aggregates in neurons. Based on that, we intoxicated mouse primary neurons and human-derived RAB39B-knock out (KO) neurons with α-Syn preformed-fibrils (PFFs) to stress the autophagic system response, finding significant alteration in LC3 and P62 canonical markers. Additionally, to evaluate α-Syn spreading, aggregation and typical DA neurodegeneration, we inject PFFs directly in RAB39b-KO mice brain. Also, due to its synaptic role enrichment, we focused on presynaptic system alteration in RAB39b-KO mice. The absence of RAB39b may alter the presynaptic vesicle formation, function and recycling with morphological and density synaptic vesicles alteration, leading to profound reduction in dopamine striatal release. We found concomitant DA neurodegeneration and loss of VMAT2 protein level in age dependent fashion in RAB39b-KO mice. In conclusion, our results demonstrate a strong interaction between RAB39B loss of function in early-onset PD development with α-Syn accumulation and DA neurodegeneration both in vitro and in vivo. Our work focus on prospectively biological interactor associations, combining mouse and human data, providing a comprehensive assessment of the disease phenotype.
Le proteine Rab, che lavorano come controllori essenziali della dinamica della membrana cellulare, recentemente associate a diversi percorsi patologici della malattia di Parkinson (MdP). In particolare, le varianti patogene di RAB39B causano disabilità intellettiva e MdP ad esordio precoce legato all'X con patologia cerebrale α-Sinucleina (α-Syn). In particolare, il gene RAB39B è specifico per il cervello, altamente espresso nei neuroni coinvolti principalmente nel traffico di vescicole intracellulari delle vie secretorie dell'ER-Golgi-endosoma, ma è stato scoperto che lavora nella formazione e nel mantenimento delle sinapsi. L'obiettivo generale di questo lavoro è trovare nuove intuizioni sul suo ruolo sconosciuto nella patogenesi del MdP e quale meccanismo potrebbe portare alla debolezza neuronale nel deficit autofagico di aggregati cellulari tossici e nell'alterazione dopaminergica presinaptica (DA). Al fine di identificare gli effettori RAB39B e le funzioni molecolari chiave a valle, il suo interactoma è stato identificato grazie all'implementazione del sistema di etichettatura APEX2 direttamente sulla coltura neuronale vivente. Attraverso questo approccio biochimico, stabiliamo un nuovo legame tra RAB39B e i complessi retromeri, con conseguenze funzionali sullo smistamento del carico come per M6PR, ATG9 o ATG9. Studiando i suoi interattori, ci siamo concentrati sulla funzione di traffico vescicolare e di membrana durante la formazione dell'autofagosoma nascente. Abbiamo ipotizzato che la perdita di funzione di RAB39B potrebbe portare a un difetto nella degradazione autofagica degli aggregati α-Syn nei neuroni. Sulla base di ciò, abbiamo intossicato neuroni primari di topo e neuroni RAB39B-knock out (KO) di derivazione umana con fibrille preformate α-Syn (PFF) per sottolineare la risposta del sistema autofagico, trovando alterazioni significative nei marcatori canonici LC3 e P62. Inoltre, per valutare la diffusione di α-Syn, l'aggregazione e la tipica neurodegenerazione DA, iniettiamo PFFs direttamente nel cervello dei topi RAB39b-KO. Inoltre, a causa del suo arricchimento del ruolo sinaptico, ci siamo concentrati sull'alterazione del sistema presinaptico nei topi RAB39b-KO. L'assenza di RAB39B può alterare la formazione, la funzione e il riciclo delle vescicole presinaptiche con alterazioni morfologiche e di densità delle vescicole sinaptiche, portando a una profonda riduzione del rilascio di dopamina striatale. Abbiamo trovato neurodegenerazione DA concomitante e perdita del livello di proteina VMAT2 in modo dipendente dall'età nei topi RAB39b-KO. In conclusione, i nostri risultati dimostrano una forte interazione tra la perdita di funzione di RAB39B nello sviluppo precoce del MdP con l'accumulo di α-Syn e la neurodegenerazione di DA sia in vitro che in vivo. Il nostro lavoro si concentra su associazioni prospettiche di interattori biologici, combinando dati murini e umani, fornendo una valutazione completa del fenotipo della malattia.

Les maladies neurodégénératives, comme Parkinson ou Alzheimer, sont une menace croissante,avec une prévalence augmentant sans cesse. Ces maladies sont caractérisées par la présence de dépôts protéiques, appellés amyloïdes, dans le cerveau. Plusieurs protéines ont été identifiées dans ces dépôts comme étant des marqueurs de la maladie, dont l'alpha-synucléine pour Parkinson et tau pour Alzheimer. L'agrégation amyloïde est centrale dans les maladies neurodégénératives et constitue ainsi un cible privilégiée pour le diagnostic ou les essais cliniques.L'agrégation amyloïde est caractérisée par la formation d'un motif cross-β, qui consiste en un empilement de brins beta;, pouvant former ainsi de longues fibres. Dans certaines conditions,des particules de taille micrométrique peuvent être obtenus, tels que les 'particulates' ou les 'sphérulites'. Plusieurs études montrent que la formation des agrégats, en particulier au stade précoce,est impliquée dans la toxicité. En revanche, les raisons de l'agrégation des protéines ne sont pas bien comprises. Dans ce travail, nous avons cherché à comprendre les principes fondamentaux impliqués dans l'agrégation amyloïde, en étudiant les changements de dynamique du système protéine-solvent, ce qui de plus, pourrait aider le développement de nouvelles méthodes de diagnostic.Dans ce but, j'ai utilisé principalement la diffusion incohérente des neutrons et les simulations de dynamique moléculaire. La première fournit une dynamique moyennée sur l'ensemble des atomes d'hydrogène dans le système et la seconde fournit une vision atomique dans laquelle structure et dynamique peuvent être étudiées.En étudiant l'alpha-synucléine, j'ai montré que les mouvements des chaines latérales et principales - dynamique interne - sont inchangés par l'agrégation. Cependant, les mouvements de l'eau sont accélérés autour des fibres, ce qui provient d'une fraction de l'eau étant déplacée du coeur hydrophobe vers les régions terminales hydrophiles lors de la formation des fibres.Ainsi, l'entropie de l'eau est augmentée dans les fibres, ou le motif cross-beta; central semble être très efficace pour se protéger lui-même de l'intéraction avec le solvent. La comparaison de la gammaS-crystalline sauvage avec le mutant G18V montre que le mutant est moins dynamique, quel que soit l'état d'agrégation. Cette observation, et la comparaison de la dynamique interne avec l'hydrophobicité des protéines, montre que la dynamique interne dépend fortement de la composition en acides aminés et non pas de l'état d'agrégation. En outre,les ions métalliques peuvent aussi influencer la dynamique interne.Les mesures sur l'insuline, en présence ou absence de zinc montre que le métal aide à l'hydratation de la protéine, même à pH 1.8, où il interagit faiblement avec la protéine. Le zinc affecte aussi les interactions entre agrégats, probablement par écrantage électrostatique, étant donné que la formation de sphérulites est facilitée en son absence.Enfin, la possibilité de suivre en simultané, et sans ambiguïté, la dynamique interne et la diffusion du centre de masse a été démontrée en utilisant des scans à fenêtre d'énergie fixe sur l'instrument IN16B à l'ILL. Cette nouvelle méthode, appliquée au lysozyme, montre que la formation des 'particulates' se déroule en une étape, avec la dynamique interne restant constante tout au long du processus. Cette expérience pilote ouvre la voie à des études de fibrillation de protéines ayant un intérêt médical.Ensemble, ces résultats démontrent que l'on peut étudier le processus d'agrégation amyloïde avec beaucoup de détails, et il y a une grande opportunité d'étendre ce travail dans un contexte biologique afin de faire le lien entre les paramètres biophysiques de l'agrégation amyloïde et ses effets et sa toxicité in-vivo
Neurodegenerative diseases, such as Parkinson's and Alzheimer's, constitute a growingthreat with ever increasing prevalence. These diseases are characterized by the presence ofprotein deposits in the patient's brain that are called amyloids. Several proteins wereidentified in these deposits as being the molecular hallmark of the disorder, among whichwe can cite alpha-synuclein for Parkinson’s disease and tau for Alzheimers’s disease.Protein amyloid aggregation is central to neurodegenerative diseases and hence constitutesa target of choice for diagnostic and therapeutic attempts. Itis characterized by the formation of a structural cross-beta pattern, which is a stack ofbeta-sheets, usually forming long fibrils. Under specific conditions, larger aggregates can beobtained, such as micrometer-sized particles, including so-called particulates andspherulites. Several pieces of evidence suggest that the formation of such aggregates, and especially at early-stages, can be involved in protein toxicity. Yet, the reasons for theaggregation to occur are not well understood. In this work, we aimed at deciphering thefundamental principles underlying protein amyloid aggregation by studying the changesin protein ad hydration water dynamics, the understanding of which might help in the development ofwater-dynamics based diagnostic methods.We employed mainly incoherent neutron scattering (on SPHERES at the MLZ and IN16B at the ILL)and molecular dynamics simulations. Theformer provides ensemble averaged information on hydrogen motions in the system, and thelatter provides a fully atomistic picture from which dynamical and structural aspects canbe investigated.Studying alpha-synuclein, we could show that protein backbone and side-chain motions - that is,internal dynamics - is barely affected by aggregation. However, hydration water motions areincreased around amyloid fibrils. The increased dynamics originates from a fraction ofwater molecules being displaced from the protein hydrophobic core to the hydrophilictermini regions when fibrils are formed. Hence, it results in a higher water entropy in fibrils,where the central cross-beta pattern appears highly efficient in protecting itself frominteracting with the solvent.For gammaS-crystallin, comparison of the internal protein dynamics of the wild-type proteinwith a G18V mutant revealed that the mutant is less dynamic, whatever itsaggregation state. This observation, along with the comparison of protein dynamics withtheir relative hydropathy index, indicates that the internal dynamics depends strongly onthe amino acid composition, but not on the aggregation state. In addition, other factorscan affect protein dynamics, such as the presence of metal ions.The measurements carriedout on insulin, in the presence or absence of zinc show that the metal promotes proteinhydration at pH 1.8, where it interacts loosely with the protein. The zincaffects also aggregate-aggregate interaction, probably by electrostatic screening as theformation of spherulites is facilitated in the absence of the metal.Eventually, the possibility to unambiguously and simultaneously access internal dynamicsand center-of-mass diffusion was demonstrated by carrying out so-called fixed- window scanson the IN16B instrument at the ILL. This novel technique applied to lysozyme showed thatparticulate formation occurs in a one-step process, and the internal dynamics remainsconstant all along. This pilot experiment opens up the possibility to study fibrilformation of pathologically relevant proteins.Taken together, the aforementioned results demonstrate that we can now study the amyloidaggregation process with great detail, and there is a great opportunity to extend this workwithin a biological context, in order to link the biophysical properties of protein amyloidaggregation with its effects and toxicity in-vivo

Mon projet de thèse a porté sur les mécanismes neurodégénératifs dans le contexte de la maladie de Parkinson (MP). Cette maladie est caractérisée notamment par la présence d’inclusions intracytoplasmiques appelées corps de Lewy, dont le composant protéique principal est l’α-synucléine. L’absence de traitements curatifs à ce jour renforce la nécessité de comprendre les processus neurodégénératifs. L’objectif de mon travail de thèse fut de proposer une approche multifactorielle, translationnelle, basée sur trois axes complémentaires: modélisation, thérapeutique et mécanistique. Premièrement, nous nous sommes intéressés à la modélisation de la MP par l’utilisation de vecteurs viraux. Cette première partie nous a permis de conclure que le vieillissement ne constitue pas un facteur de risque pour les trois espèces étudiées. Ensuite, nous avons étudié deux stratégies pour combattre la dysfonction lysosomale existant chez les patients, premièrement par une approche biotechnologique avec des nanoparticules permettant de restaurer le pH des lysosomes dysfonctionnels, et une stratégie de thérapie génique par surexpression d’un régulateur de la biogénèse lysosomale. Grâce à ce travail, nous avons démontré l’intérêt du lysosome comme cible thérapeutique. Enfin, nous nous sommes focalisés sur l’hypothèse « prion » pour les synucléinopathies. Dans ce projet, nous avons mis en œuvre une approche de modélisation chez le primate non-humain ainsi qu’une une approche thérapeutique anti-agrégative chez le rongeur. Ces travaux mettent en évidence le rôle clé de l’α-synucléine dans l’étiologie de la MP et proposent des pistes d’améliorations des modèles animaux actuels ainsi que des approches thérapeutiques innovantes
The aim of this work was to focus on neurodegenerative mechanisms in the context of synucleinopathies, especially on Parkinson’s disease (PD). PD is characterized by the loss of dopaminergic neurons and the presence of intracytoplasmic proteinaceous inclusions named Lewy Bodies of which α-synuclein (α-syn) is the main protein component. To date, there are no curative treatments. Elucidating mechanisms underlying neurodegeneration in PD will allow the identification of new molecular targets for therapeutic intervention. My Ph.D. work intends multifactorial and translational approaches based on modelling, therapeutic intervention and mechanistic studies. We first focused on the development of new animal models of PD based on the use of viral vector-mediated overexpression of α-syn. This word allowed us to conclude on the absence of additive effect of ageing in α-syn-related toxicity, at least in the three investigated species. Then, we worked on two therapeutic strategies to overcome the lysosomal dysfunction occurring in PD. To do so, we first developed a biotechnological approach based on the use of acidic nanoparticles restoring acidic pH of sick lysosomes, and then we used a gene therapy approach based on the overexpression on a central modulator lysosomal biogenesis. We here demonstrated the interest of restoration of lysosomal physiology. Finally, we tested the “prion-like” hypothesis in a cohort of nonhuman primates and assessed the efficacy of a therapeutic approach using an oligomer modulator in mice. This work highlights the central role of α-syn in PD etiology and offers innovative strategies for both modelling and therapeutic intervention

The main goal of this research is to provide a novel bioanalytical approach to better understand α-synuclein (AS) aggregation linked to Parkinson’s disease (PD) and characterize the implications of contributing factors such as the presence of metal ions and potential therapeutics that would inhibit or reverse AS fibrillation. Current bioanalytical techniques have reported the fibrillation process of AS however, the detection of prefibrillar formation or the nucleation phase of AS has yet to be characterized. This research aimed to address this issue and monitor the primary stages of AS fibrillation from natively soluble monomer to fibrillar aggregates. The electrochemical measurement of these processes utilized the intrinsic electroactivity of 4 tyrosine (Tyr) residues in AS observed at ~0.6 V (vs. Ag/AgCl) to monitor its early fibrillation kinetics. The research presented here provided valuable evidence of the conformational changes attributed to prefibrillar forms of AS.

Tese de mestrado. Biologia (Biologia Humana e Ambiente), Universidade de Lisboa, Faculdade de Ciências, 2012
O envelhecimento da população humana está associado ao aumento da incidência de doenças neurodegenerativas, como a doença de Parkinson (DP) e a doença de Alzheimer. O misfolding e subsequente agregação de proteínas é uma característica patológica presente em diferentes doenças neurodegenerativas. No entanto, permanece por esclarecer se tais eventos se tratam de uma causa ou uma consequência da progressão da doença. O primeiro gene implicado na DP foi SNCA que codifica a proteína alfa-sinucleína (aSyn), principal proteína que compõe os corpos de Lewy (LB), agregados proteicos que se acumulam nos neurónios dos pacientes com DP. É igualmente conhecido que a sobre expressão da aSyn em vários modelos animais com DP resulta em citotoxicidade. A maioria dos casos DP são esporádicos, no entanto cerca de ~5-10% de casos familiares estão ligados a mutações específicas em diferentes genes. Até ao momento, são conhecidas três mutações no gene que codifica aSyn e que se encontram igualmente associado à etiologia dos casos familiares de DP. No entanto, os mecanismos pelos quais cada mutação leva à doença, são ainda desconhecidos. O objetivo deste projeto é investigar o efeito das diferentes mutações na formação de inclusões de aSyn ligada aos casos familiares (A30P, E4K e A53T), bem como mutações artificiais, conhecidas por interferirem com a biologia da agregação de aSyn (S129A, S129D, S87A, S87E, tripla prolina, Y125). Espera-se que esta análise comparativa forneça informações importantes sobre os mecanismos moleculares envolvidos no processo agregação/misfolding da aSyn, permitindo assim o desenvolvimento de novas e estratégias de intervenção na DP e outras sinucleinopatias.
The aging of the human population is associated with an increased incidence of neurodegenerative diseases, such as Parkinson’s disease (PD) and Alzheimer’s disease (AD). Protein misfolding and aggregation is one of the pathological hallmarks present in different neurodegenerative disorders, but it is still unclear whether this is a cause or a consequence of the disease progression. The first gene implicated in PD was SNCA. This gene encodes alpha-synuclein protein (aSyn), which is the main protein component of Lewy bodies (LB). LBs are protein inclusions that accumulate in living neurons of PD patients. It is known that, when overexpressed, aSyn results in an increase of cytotoxicity, in several cell-based and animal models of PD. The majority of PD cases are sporadic and only ~5-10% are linked to familial cases and are associated with a specific gene mutation. To date, three mutations in the gene encoding aSyn are known, suggesting that it also plays a major role in the etiology of familial cases of PD. Nevertheless, the mechanisms through which each mutation leads to disease, are still not known. The purpose of this study is to investigate the effect of inclusion formation of different familial aSyn mutations (A30P, E4K and A53T), as well as artificial mutations that are known to interfere with the normal biology and aggregation of the protein (S129A, S129D, S87A, S87E, Triple proline mutation, Y125). Ultimately, this comparative analysis will provide important insight into the molecular mechanisms involved in the misfolding/aggregation process and may enable the development of novel strategies, for intervention in PD and other synucleinopathies.

碩士
國立臺灣師範大學
生命科學研究所
102
Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting people in their middle and old age. It is characterized by resting tremor, rigidity, akinesia and postural instability and associated with selected loss of dopaminergic (DA) neuron in the substantianigra pars compacta. Combinations of environmental and genetic factors are thought to cause PD. Among the genetic factors contributing to the pathogenesis of PD, mutations in the α-synuclein gene such as A30P, A53T and E46K increased aggregate formation and mutations in Parkin, DJ-1 and PINK1 impaired mitochondrial function. In addition, environmental factors as well as aging are also thought to contribute the development of the disease. No effective treatment for the disease currently urges the development of new agents that may halt the degeneration of PD. In this study, human neuroblastoma SH-SY5Y cells were differentiated toward the DA phenotype in the presence of retinoic acid (RA) follow by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Greatly up-regulated tyrosine hydroxylase (TH, DA neuronal marker) in SH-SY5Y cells was observed following 7 days of differentiation. These TH-positive cells exhibited a mature morphology with long, branched cell processes and large cell bodies. Addition of MPP+ (1-methyl-4-phenylpyridinium), a potent complex I inhibitor in DA neurons, to the above SH-SY5Y cells caused significant cell death compared with untreated cells. This MPP+-induced cell death was used to screen herbal extracts provided by Industrial Technology Research Institute (ITRI). Cell viability assay indicated that pretreatment of herbal extracts NTNU-043, -057, -059, -125, -293, -313, -331, -385, -450, and -514 protected undifferentiated SH-SY5Y cells against MPP+-induced toxicity, whereas pretreatment of herbal extracts NTNU-092 and -313 protected differentiated SH-SY5Y cells against MPP+-induced toxicity.

α-Synucleinopathies are a vast group of neurodegenerative disorders characterized by the abnormal accumulation of insoluble aggregates, both in neurons and in oligodendrocytes, whose major component is the protein α-synuclein (αS). Among them, Parkinson’s disease (PD) is the most widespread; it is defined by the progressive loss of dopaminergic neurons in the substantia nigra, responsible for several motor disturbances, such as bradykinesia, muscular rigidity and resting tremor. This work is focused on αS, whose abnormal self-assembly gives rise to insoluble inclusions called Lewy bodies and neurites, the most relevant neuropathological hallmarks of PD. The aggregation process of αS is extremely complex and leads to the formation of a wide range of assemblies such as oligomers, protofibrils and fibrils. To define the nature of the species responsible for neuronal damage and their mechanism of action, in the first part of this work we have evaluated the evolution in time of different readouts of cellular dysfunction in neuronal cells. We found that, at early incubation times, small oligomeric species with a rudimentary cross-β structure and high solvent exposed hydrophobicity are by far the most toxic to cells, whereas unstructured monomers and hydrophilic and disordered oligomers are unable to cause any cellular dysfunction. We also found that αS fibrils induce the same cascade of events as toxic oligomers, but more slowly and at a rate dependent on their length, despite their inability to be internalized by the cells. Thus, we associated the toxic capacity of αS fibrillar assemblies with their ability to release small hydrophobic oligomers with a cross-β architecture, particularly effective in crossing neuronal membranes and in inducing neurotoxicity. Our results indicate that oligomers are the most toxic among the analyzed αS species, but fibrillar assemblies can generate neurotoxicity through the release of small oligomeric aggregates, that can in turn contribute to the toxicity associated with their well-characterized ability to transfer from neuron-to-neuron, causing the spreading of Lewy body pathology. In the second part of this study we focused on the ability of αS species to interact with neuronal membranes and we analyzed the involvement of the different membrane components, in particular the exposed proteins, on this interaction. Our study revealed that αS oligomers accumulate on the plasma membrane in close proximity to the cellular prion protein, subsequently inducing an increase of intracellular calcium influx in cells by both channel-independent and channel-dependent mechanisms, with the N-methyl-D-aspartate receptor-channels (NMDARs) triggering a prompt and transient calcium influx, followed by a massive calcium dysregulation due to the disruption of the plasma membrane integrity. Accordingly, the pharmacological inhibition of NMDARs, as well as the blockade of the cellular prion protein and the removal of the proteins exposed on the cell membrane transiently delayed the early calcium influx, but not the sustained late one caused by αS oligomers. Furthermore, αS fibrils caused calcium dyshomeostasis with slower kinetics with respect to the oligomers, and the observed ionic alterations were not rescued by the blockade of NMDARs or by the removal of the proteins exposed in neuronal membranes. Thus, the experimental evidences accumulated in the second part of this work shed light into the interplay between αS aggregates and the plasma membrane of neuronal cells, thus expanding the range of molecular targets for the therapeutic intervention in PD. Overall, the data presented in this work provide a robust body of evidence on the prominent role of oligomeric species with high solvent exposed hydrophobicity and cross-β structure, formed either during the aggregation process of αS, or released from mature fibrils, in the neurotoxicity of αS, giving a detailed description of the toxic effects they evoke. The experimental evidences accumulated in this study also emphasize the importance of the membrane binding properties of such species for their pathological features, proposing possible strategies with therapeutic value in PD.

9 ABSTRACT For proper function proteins should have a native conformation. If their conformation is impaired due to environmental stress or genetic mutation, proteins become prone to aggregation. There exist various types of protein aggregates. Stable non-membraneous inclusions can form which can serve for clearance of aberrant proteins from place where they can interfere with essential cellular processes. Another type of aggregates can serve as transient deposits of proteins thus protecting them from stress conditions. Stress granules (SG) are a such example of transient granules. Their formation is induced by heat shock for example. SGs contain mRNA, components of translation machinery, and other proteins. One of these proteins is Mmi1, small highly conserved protein with unknown function. Association of Mmi1 with stress granules and partial co-localization with chaperon Cdc48 and proteasom indicates Mmi1 can mediate heat stress damaged protein degradation. We have uncovered that yeast prion protein Sup35 is a component of stress granules as well. With regard to its aggregation capability there existed an assumption that prion domain of Sup35 could serve as scaffold for SG assembly. However as we show deletion of prion domain of Sup35 protein does not affect stress granules formation dynamics. Yeast...