Dissertations / Theses on the topic 'Alpha-Synuclein, Prion Protein, Aggregation'

Abstract: Neurodegenerative diseases are associated with progressive loss of structure or function of neurons which results in cell death. Recent evidence indicate that all neurodegenerative disorders, sporadic or transmissible, may have a common pathological mechanism at the molecular level. This common feature consists of protein aggregation and accumulation of harmful aggregates in neuronal cells resulting in cellular apoptosis and neurotoxicity. Neurodegenerative diseases can affect abstract thinking, skilled movements, emotional feelings, cognition, memory and other abilities. This diverse group of diseases includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), prion diseases or transmissible spongiform encephalopathies (TSEs) and amyotrophic lateral sclerosis. In my project I worked on the molecular mechanism of protein aggregation, propagation and neurotoxicity in Parkinson's disease and prion disease. Prion disease and PD are associated with misfolding and aggregation of PrPc and a-Synuclein (a-Syn), respectively. Despite being two important neurodegenerative disorders, molecular mechanisms of a-Syn or PrPC aggregation and amyloidogenesis are still unclear in PD and prion disease. Furthermore, the toxic protein species in PD have not been characterized yet. In this study we characterize the mechanism of a-Syn and PrPc misfolding in a physiological-like cell free condition in the absence of a-Syn aggregates, PrPc ggregated isoform (Pre's), denaturants or acidic environment. A number of studies indicate that dimerization of PrPc or a-Syn may be a key step in the aggregation process. To test this hypothesis we verified if enforced dimerization of PrPc or a-Syn may induce a conformational change reminiscent of the conversion of PrPc or a-Syn to PrPR' or a-Syn aggregates, respectively. We used a well-described inducible dimerization strategy where a dimerizing domain called FK506-binding protein (Fv) was fused to PrPc or a-Syn in order to produce chimeric proteins Fv-PrP and a-SynF'''. A divalent ligand AP20187 was used to induce protein dimerization. Addition of AP20187 to recombinant Fv-PrP in physiological-like conditions resulted in a rapid conformational change characterized by an increase in beta-sheet (13-Sheet) structure and simultaneous aggregation of the proteins. However, non-dimerized PrP formed 13-Sheet conformation in very slower rates. In the presence of AP20187, we also report a rapid random coil into 13-sheet conformational transformation of a-SynF" within 24 h, whereas wild type a-Syn showed 24 h delay to achieve P-sheet structure after 48 h. Electron microscopy experiments demonstrated that dimerization induced amyloid fibril formation after 48 h for both Fv-PrP and a-Syr?", whereas in the absence of dimerizing ligand AP20187, PrP or a-Syn converted into amyloid fibrils after 3 days or even later. Dimerization-induced Fv-PrP aggregates were partially resistant to PK digestion which is a characteristics of the naturally occurring PrPR'. The rates of amyloidogenesis in the presence of dimerization was also characterized by Thioflavin T (ThT) fluorescence probing. Whereas the stable structure of Fv-PrP showed no ThT binding for over 60 h of incubation at 37°C, the addition of AP20187 to Fv-PrP resulted in a time-dependent increase in ThT binding. As for a-SynR, dimerization accelerated the rate of ThT binding and amyloid formation comparing to the slower amyloidogenesis rate of wild type a-Syn in the absence of dimerizer AP20187. The impact of dimerization on a-Syn aggregation was further determined by Fluorescence ANS probing, indicating a higher affinity of dimerization-induced a-SynF" aggregates for binding to ANS comparing to wild type a-Syn aggregates. These results indicate that dimerization increases the aggregation and amyloidogenesis processes for Fv-PrP and a-SynF". Both Fv-PrP and a-SynF" amyloids were successfully propagated in vitro by protein misfolding amplification (PMCA) cycle. These results ar in agreement with the theory that all protein aggregates in neurodegenerative diseases propagate with the same molecular mechanism. Neurotoxicity of recombinant Fv-PrP and a-SynF" aggregates was determined in cellulo and in vivo, respectively. Aggregates of Fv-PrP were toxic to cultured cells whilst soluble Fv-PrP and amyloid fibres were harmless to the cells. When injected to the mice brain, both a-Syni" and a-Syn pre-fibrillar aggregates internalized cells and induced neurotoxicity in the hippocampus of wild-type mice. These recombinant toxic aggregates further converted into non-toxic amyloids which were successfully amplified by PMCA method, providing the first evidence for the in vitro propagation of synthetic a-Syn aggregates. These results suggest an important role for protein dimerization in aggregation and amyloidogenesis, and therefore, in the pathology of PD and prion disease. The similarities between aggregation, amyloidogenesis and toxicity of PrPC and ct-Syn provide further evidence on the existance of a prion-like mechanism in all neurodegenerative disorders. // Résumé: Les maladies neurodégénératives sont associées à la perte progressive des propriétés structurales ou fonctionnelles des neurones, ce qui engendre la mort des cellules. De récentes études indiquent que tous les désordres neurodégénératifs, sporadiques ou transmissibles, peuvent avoir un mécanisme pathologique commun au niveau moléculaire. Ce dispositif commun se compose de l'agrégation de protéines, de la propagation des agrégats, et de l'accumulation d’agrégats toxiques dans les cellules neuronales, menant à l'apoptose et à la neurotoxicité cellulaire. Les maladies neurodégénératives peuvent affecter la pensée abstraite, les mouvements habiles, les sentiments émotifs, la connaissance, la Mémoire et d'autres capacités cognitives. Ce groupe divers de maladies inclut la maladie d'Alzheimer (AD), de Parkinson (PD), de Huntington (HD), les maladies à prions ou encéphalopathies spongiformes transmissibles (TSEs) et la sclérose latérale amyotrophique (ALS). [symboles non conformes]

The pathological hallmark of many age-related neurodegenerative diseases is the presence of proteinaceous inclusions in nerve cells and glial cells. Alpha-synuclein is the main component of the inclusions of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy, as well as of rarer diseases, collectively called synucleinopathies. For a long time, it was widely believed that neurodegenerative diseases were cell-autonomous; however, a more recent hypothesis has suggested that some misfolded proteins resemble prions. Thus, aggregated alpha-synuclein shares features of PrPSc, the scrapie form of the prion protein. The aim of this thesis was to further characterize the prion-like properties of aggregated alpha-synuclein by studying the pathways of seeded aggregation, and to identify the species of alpha-synuclein responsible. I present evidence, using a HEK 293T cell model, that filamentous protein was the most seed-potent form of alpha-synuclein. Recombinant aggregated protein, aggregated alpha-synuclein from mice transgenic for A53T alpha-synuclein, as well as alpha-synuclein aggregates from Parkinson’s disease and multiple system atrophy brains, seeded aggregation. The mechanisms of alpha-synuclein internalization and intracellular trafficking, and how these processes affect seeded aggregation, are not fully understood. I showed that internalization of alpha-synuclein aggregates occurs through clathrin- and dynamin-independent, Cdc42-, actin- and PI3K-dependent endocytosis. Alpha-synuclein aggregates are trafficked to the endolysosomal pathway; a small fraction of lysosomes ruptures, which induces aggregation of expressed cytoplasmic alpha-synuclein, and disruption of autophagy, which in turn enhances seeded aggregation. These findings expand knowledge of the prion-like properties of assembled alpha-synuclein and identify novel mechanisms with therapeutic potential.

The aggregation of alpha-synuclein (αS) protein from soluble monomer into solid amyloid fibrils in the brain is associated with a range of devastating neurodegenerative disorders such as Parkinson’s disease. Soluble oligomers formed during the aggregation process are highly neurotoxic and are thought to play a key role in the onset and spreading of disease. Despite their importance, these species are difficult to study by conventional experimental approaches owing to their transient nature, heterogeneity, low abundance and a remarkable sensitivity of the oligomerisation process to the chosen experimental conditions. In this thesis, well-established single-molecule techniques have been utilised to study the aggregation and oligomerisation of αS in solution.

Parkinson’s disease (PD) is the most important neurodegenerative disease which regards movement. The 1% of the population over 65 years old is affected by this disorder. The main symptoms are bradykinesia, resting tremor, postural instability, muscle rigidity and sometimes cognitive and personality problems. The cause of the disease is a selective death of dopaminergic neurons in substantia nigra pars compacta. Actually, the best therapy can help to solve only symptoms and it is based on the supply of the precursor of dopamine, which is the neurotransmitter lacking in the disease, or inhibitors of the activity of enzymes involved in the metabolism of dopamine. This therapy does not prevent further neuronal loss. Two are the links that correlate the protein alpha-synuclein (?-syn) to PD: this protein is found as amyloid fibrils in proteinaceous aggregates known as Lewy bodies, which are present in PD patients’ brains, and second, single point mutation of ?-syn are correlated to early onset of autosomic dominant forms of the disease. In this frame an understanding the molecular cause that lead to neuronal loss and protein aggregation becomes crucial for the development of new therapeutic strategies. ?-Syn is expressed in all the central nervous system and it is localized at the presynaptic terminal but its biological role is still not clear. ?-Syn is natively unfolded and it is able to acquire different conformations in different conditions such as the presence of membranes or organic solvents. The central region of the protein is able to fold into ?-sheet structure comparable with amyloid fibrils found in Lewy bodies. Point mutants implied in the early onset PD (A30P, E46K and A53T) have a higher propensity for the formation of oligomers. Recently, the hypothesis that the oligomers are the main cause of ?-syn toxicity is gaining support. Studying the oligomerization process seem to be now more important for the comprehension of neuronal death. The first steps of self-interaction are extremely rare events and thus difficult to observe with bulk methods; fibrils are insoluble, so structure can not be solved by NMR, nor by X-ray crystallography. Moreover, ?-syn was found to interact with a wide variety of proteins as detected by co-immunoprecipitation or affinity techniques. The biological relevance and the molecular basis of this processes require further investigation by high resolution methods like NMR (Nuclear Magnetic Resonance) or SPR (Surface Plasmon Resonance). Furthermore, every interacting partners may sequester ?-syn from cytosol to decrease the probability of self-interaction that lead to aggregation. In this PhD thesis investigations were done in order to improve ?-syn interaction network knowledge. As any event correlated with an altered balance of ?-syn interaction network may favour ?-syn self-interaction, the experimental approach was divided into three parts to get information about: protein-protein interaction, membrane binding and aggregation studies. SPR studies was performed to verify the interaction between ?-syn and 14-3-3?. 14-3-3 chaperone family can bind and regulate a wide variety of proteins. Sato et al. (2006) measured 1.1 ?M dissociation constant between ?-syn and 14-3-3? by SPR. However, these data were not reproduced, and also HSQC spectra of 15N labelled ?-syn in the presence of a three molar excess of 14-3-3? did not provide evidences of an interaction between the two molecules. Interaction between membranes and ?-syn was studied by circular dichroism (CD). The first hundred residues of the proteins acquire ?-helix structure upon binding with micelles and liposomes. Interesting data come from the interaction between ?-syn dimers formed by two mutants produced in our lab (V3C and Syn141C): the dimer formed by disulfide bond between the Cys at the C-terminal end of the protein (C-term dimer) forms a distorted ?-helix upon the binding with 50 nm diameter small unilamellar vesicles (SUVs) composed of 50% DMPG 50% DMPC, while the dimer formed by V3C mutants (N-term dimer) acquires an amount of ?-helix comparable to the one observed upon binding to SDS micelles. It is possible that SUV dimensions (i.e. curvature) and the covalent constrain in C-term dimer are the cause of helix distortion. Finally, self-interaction of ?-syn was investigated by fibrillogenesis and aggregation assays. Fibrillogenesis was monitored with Thioflavin T (ThT) fluorescence; samples of wild-type ?-syn, C-term dimer, pathological mutant A30P, E46K and A53T were incubated at 37°C under shaking; aliquots were collected at fixed time, mixed with ThT solution and fluorescence intensity measured at 485 nm. This assay revealed that E46K, A53T and C-term dimer form fibrils faster than wild-type ?-syn and the A30P mutant presents a longer lag phase. It was not possible to obtain good sigmoidal curves with this method in the case of ?-syn, probably because of ?-syn fibrils disruption or precipitation and light scattering events. Hence, a protocol applied by Lük et al. (2007) was applied. This method measures fluorescence polarization (FP) of samples of ?-syn incubated in a 96 wells plate at 37°C under agitation. ?-Syn wild type protein, pathological mutants, C-term and N-term dimer were mixed with Oregon Green 488 maleimide labelled ?-syn (1:100=Syn-OregonGreen:?-syn), to then measured FP variations in time. The comparison between the samples shows that wild-type ?-syn aggregates faster than pathological mutants and N-term dimer. The C-term dimer shows an increase of FP with the shortest lag phase. The covalent constrain seem to favour intramolecular interaction and then aggregation and fibrillogenesis. NMR spectra was recorded for C-term dimer formed with 1:5 protein mixture of 15N labelled Cys C-term mutant : 14N Cys C-term mutant, but no intramolecular interaction was detected. In addition, ?-syn was tested in the presence of three proteins. While DJ1 provides no significance effect on ?-syn aggregation, 3T protein seem to have an aspecific influence on oligomers enlargement rate. Moreover, 14-3-3? mixed in three molar ratios to ?-syn seems to have a concentration dependent effect on ?-syn aggregation, although experimental errors do not allow a conclusive interpretation of this finding. However, 1:1=14-3-3?:?-syn shows significantly slower aggregation rate compared to ?-syn incubated alone. In conclusion, progress on the understanding on the molecular mechanism of ?-syn aggregation was reached, specifically for what concern the orientation of intramolecular interaction that lead to the formation of oligomers and fibrils, and proteins able to host ?-syn oligomers growth. Moreover, a new method based on fluorescence polarization was used to reveal differences on lag phase and rate of the aggregation process of ?-syn and its variants. This technique can be use to test conditions, molecules and proteins able affect the aggregation of ?-syn.
Il morbo di Parkinson (PD) è la più importante malattia neurodegenerativa riguardante la funzionalità motoria. L'1% della popolazione sopra i 65 anni è affetto da questa malattia. I sintomi principali sono bradichinesia, tremore a riposo, instabilità posturale, rigidità muscolare e, talvolta, problemi cognitivi e della personalità. La causa della malattia è una morte selettiva dei neuroni dopaminergici nella substantia nigra pars compacta. In realtà, la migliore terapia attualmente applicata è puramente sintomatica, e si basa sulla somministrazione del precursore della dopamina, che è il neurotrasmettitore assente nella malattia, o su inibitori delle attività degli enzimi coinvolti nel metabolismo della dopamina. Questa terapia non impedisce un’ulteriore perdita neuronale. Due evidenze correlano la proteina alfa-sinucleina (?-syn) al PD: questa proteina è presente come fibrille amiloidi in aggregati proteici noti come corpi di Lewy, che sono presenti nel cervello dei pazienti, e in secondo luogo, mutazioni di un singolo amminoacido del gene di ?-syn sono correlati all’insorgenza di forme precoci della malattia, con trasmissione autosomica dominante. In questo contesto, la comprensione delle cause molecolari che conducono alla perdita di neuroni e all’aggregazione di ?-syn diventa fondamentale per lo sviluppo di nuove strategie terapeutiche. ?-Syn è espressa in tutto il sistema nervoso centrale ed è localizzata presso i terminali presinaptici, tuttavia il suo ruolo biologico non è ancora chiaro. ?-Syn è una natively unfolded protein, ma è in grado di acquisire conformazioni diverse in diverse condizioni, quali la presenza di membrane o solventi organici. La regione centrale della proteina è in grado di acquisire strutture a foglietto ? nelle fibrille amiloidi che vengono riscontrate nei corpi di Lewy. I mutanti patologici (A30P, E46K e A53T) hanno una maggiore propensione per la formazione di oligomeri. Recentemente, si sta rafforzando l'ipotesi che gli oligomeri siano la principale causa della tossicità causata da ?-syn. Studiare il processo di oligomerizzazione è quindi di enorme importanza per la comprensione dei processi che portano alla morte neuronale. I primi passaggi nella creazione di piccoli aggregati sono eventi estremamente rari, e quindi difficili da osservare con maggior parte dei metodi; in più, essendo le fibrille insolubili, la loro struttura non può essere risolta da NMR, né dalla cristallografia a Raggi-X. Diversi studi riportano l’interazione di ?-syn con una grande varietà di proteine, come rilevato da esperimenti di co-immunoprecipitazione o cromatografia di affinità. La rilevanza biologica e la base molecolare di questo processo necessitano di un'ulteriore indagine con metodi ad alta risoluzione come NMR (Risonanza Magnetica Nucleare) o SPR (Surface Plasmon Resonance). Inoltre, tutte le macromolecole in grado di interagire con ?-syn ne provocano il sequestro dal citosol, diminuendo le probabilità di auto-interazione che portano alla sua aggregazione. In questa tesi di dottorato sono stati realizzati studi al fine di ampliare la conoscenza sulla rete di interazione di ?-syn. Dal momento che ogni evento correlato ad un alterato l'equilibrio nel network di interazioni di ?-syn può favorire la fibrillogenesi, l'approccio sperimentale è stato diviso in tre parti: interazioni proteina-proteina, legame alle membrane e studi di aggregazione. Esperimenti mediante SPR sono stati effettuati per verificare l'interazione tra ?-syn e 14-3-3?. La famiglia di chaperone 14-3-3 può interagire e regolare una grande varietà di proteine. Sato et al. (2006) hanno misurato con tecniche SPR la costante di dissociazione tra ? e syn-14-3-3?, riportando un valore di (1,1 ?M). Negli esperimenti effettuati questo dato non è stato riprodotto, e anche lo spettro HSQC di ?-syn marcata con 15N in presenza di tre volte eccesso molare di 14-3-3? non ha fornito prove di un’interazione tra le due molecole. Il legame alle membrane di ?-syn è stato studiato mediante dicroismo circolare (CD). I primi 100 residui della proteina sono in grado di acquisire struttura ?-elicoidale in presenza di micelle e liposomi carichi negativamente. Dati interessanti provengono dallo studio di dimeri di ?-syn costituiti da due mutanti prodotti nel nostro laboratorio (V3C e Syn141C): l’omodimero formato da un ponte disolfuro tra la cisteina posizionata al C-terminale della proteina (dimero C-term) forma un’?-elica distorta in presenza di liposomi di 50 nm di diametro, composti di 50% DMPG 50% DMPC. Il dimero formato dal mutante V3C (dimero N-term) acquisisce struttura ?-elicoidale paragonabile a quella osservata per il legame con micelle di SDS. È possibile che la dimensione (cioè la curvatura) dei liposomi e il legame covalente vincolante la coda C-terminale nel dimero C-term siano la causa dell’alterazione della struttura dell’?-elica. Infine, la self-interazione di ?-syn è stata oggetto di indagine con saggi di fibrillogenesi e di aggregazione. La formazione di fibrille è stata rilevata sulla base della variazione di intensità della fluorescenza della molecola Tioflavina T (ThT); campioni di wild-type ?-syn, dimero C-term e mutanti patologici A30P, E46K e A53T sono stati incubati a 37 °C sotto agitazione; aliquote sono state raccolte a tempi fissi, miscelate con una soluzione di ThT e l’intensità di fluorescenza misurata a 485 nm. Il test ha rivelato che E46K, A53T e il dimero C-term formano fibrille più velocemente rispetto a wild-type ?-syn, il mutante A30P presenta invece un ritardo nella lag-phase. Non è stato possibile ottenere una buona interpolazione dei dati con questo metodo, probabilmente a causa della precipitazione o della rottura delle fibrille di ?-syn, o di eventi di light scattering in cuvetta dovuti alle fibrille. Pertanto, un protocollo pubblicato da Luk et al. (2007) è stato applicato. Questo metodo misura l’aumento della polarizzazione di fluorescenza (FP) di campioni di ?-syn incubati a 37 °C sotto agitazione in una piastra a 96 pozzetti. ?-Syn wild-type, mutanti patologici, dimeri C-term ed N-term sono stati mescolati con ?-syn marcata con Oregon Green 488 (1:100 = Syn-OregonGreen: syn-?), e le variazioni nel tempo di FP sono state registrate. Il confronto tra i campioni dimostra che ?-syn wild-type aggrega più veloce rispetto ai mutanti patologici e al dimero N-term, mentre il dimero C-term presenta il più veloce aumento di FP, con la minor lag-phase.. Il legame covalente al C-terminale sembra favorire l'interazione intramolecolare e quindi l'aggregazione e la fibrillogenesi. Lo spettro NMR è stato registrato per il dimero C-term formato per il 20% da molecole di ?-syn marcate con 15N, ma non è stata rilevata interazione intramolecolare. Inoltre, l’aggregazione di ?-syn è stata testata in presenza di tre proteine. Mentre la presenza di DJ1 non comporta effetti statisticamente significatici sull’aggregazione di ?-syn, la proteina chimerica 3T influenza la velocità di ingrandimento degli oligomeri di ?-syn. Inoltre, il chaperone 14-3-3? mescolato in tre rapporti molari con ?-syn sembra avere un effetto concentrazione dipendente sull’aggregazione di ?-syn, anche se gli errori sperimentali non consentono una interpretazione conclusiva di questa osservazione. Tuttavia, ?-syn in presenza di 14-3-3? equimolare mostra una velocità di aggregazione significativamente più lenta rispetto ai campioni di ?-syn incubati in assenza di 14-3-3?. In conclusione, sono stati raggiunti dei progressi sulla comprensione sul meccanismo molecolare di aggregazione ?-syn, in particolare per ciò che riguarda l'orientamento dell’interazione intramolecolare che porta alla formazione di oligomeri e fibrille, e le proteine in grado di ostacolare la crescita di oligomeri di ?-syn. Inoltre, un nuovo metodo basato sulla polarizzazione di fluorescenza è stata utilizzato per rilevare differenze in velocità di aggregazione e lag phase tra ?-syn e sue varianti. Questa tecnica può essere utilizzata per testare diverse condizioni, molecole e proteine in grado di influenzare l'aggregazione in vitro di ?-syn.

Parkinson’s disease is the second most common neurodegenerative disorder after Alzheimer’s disease and affects about 1% of the population over 65 years old. This disorder can be both sporadic and familial and some genetic forms are due to mutations in SNCA gene, encoding for the protein alpha-synuclein (aS). PD pathological hallmarks are the prominent death of the dopaminergic neurons in the substantia nigra pars compacta and the presence of proteins and lipid inclusions, termed Lewy’s body (LBs), in the surviving neurons in parkinsonian brains. The main constituent of LBs is an aggregated fibrillar beta-sheet rich form of aS. aS aggregation process was widely studied in the past years: the protein is unfolded in its native state, but in pathological conditions it tends to aggregate forming oligomeric species. These oligomers constitute a heterogeneous and transient ensemble and rapidly convert into amyloid fibrils when they reach a critical concentration. Amyloid fibrils then deposit in LBs along with several other proteins and lipids. aS aggregation was mainly studied in vitro, but recently more efforts were put into the study of this process in cell and animal models, to identify not only aS aggregation intermediates, but also the associated toxic mechanism(s) that lead to neurons cell death in PD. In this thesis two main issues were faced: the study of aS aggregation in cells using unconventional methods and the characterization of the effects of the family of chaperone-like proteins 14-3-3, on aS aggregation. In the first part, two cellular models for the study of aS aggregation were set and characterized: the first one is obtained just overexpressing aS and allowed the characterization of an ensemble of heterogeneous oligomeric species (about 6±4 monomers per oligomer) using a new fluorescence microscopy method termed Number and Brightness analysis. These oligomeric species induced autophagic lysosomal pathway activation and mitochondrial fragmentation in this model. The second cellular model provides a method to study aS fibrils and larger aggregates in a physiological environment: aS was overexpressed in cells and aggregation was triggered by introducing in cell cytoplasm recombinant aS fibrils fragments, termed seeds. In both cases aS overexpression and aggregation cause cellular death, in good agreement with what was previously published by others groups. The characterization of aS aggregation in cells went further looking at the variation in cellular metabolism, possibly induced by mitochondrial damage. These changes were quantified measuring NADH fluorescence properties in the two models with respect to the control. These results showed that in cells presenting aS oligomer or aggregates, NADH fluorescence lifetime and emission spectra change, suggesting that these measurements may be used to detect aS aggregates in live cells and in vivo using a non-invasive dye-free method. The second part of the thesis concerns the ability of 14-3-3 chaperone-like proteins of interacting with aS and of interfering with aS aggregation process rescuing the induced toxicity in cells. Among the seven 14-3-3 isoforms, 14-3-3 eta can re-route aS amyloidogenic process in vitro, leading to the formation of curved objects rather than aS fibrils. These curved objects have diameters and curvatures that depend on 14-3-3 eta amount in the aggregation assays; moreover, 14-3-3 eta molecules were found in these aggregates, suggesting the formation of a stable complex between the two proteins. When aS amount is too large or seeds are used to trigger the aggregation process in vitro, 14-3-3 eta is not able any more to affect aS aggregation and is sequestered into aS fibrils. In cell models, 14-3-3 eta overexpression leads to a rescue when aS was only overexpressed, but not when aggregation in cell cytoplasm was triggered by seeds. Overexpressed 14-3-3 eta was found to interact with overexpressed aS using image correlation spectroscopy methods (cross raster image correlation spectroscopy and cross Number and Brightness analysis), mainly at plasma membrane. Moreover, 14-3-3 eta is sequestered into aggregates when aS aggregation is triggered by seeds, highlighting another possible toxic mechanism due to aS aggregation. All the results obtained in cells are in good agreement with the in vitro results previously reported, further suggesting that 14-3-3 proteins and eta isoform in particular are interesting in aS aggregation frame and may be used to interfere in the process to rescue its toxic effects.
La malattina di Parkinson è la seconda malattia neurodegenerative più comune dopo il morbo di Alzheimer e colpisce circa l’1% delle popolazione sopra i 65 anni di età. Questa malattia può essere sia sporadica che familiare e alcune forme genetiche sono dovute a mutazioni nel gene SNCA che codifica per la proteina alfa-sinucleina. Le caratteristiche patologiche principali della malattia di Parkinson sono la morte prevalentemente dei neuroni dopaminergici della substantia nigra pars compacta e la presentza di inclusioni proteiche e lipidiche, dette corpi di Lewy, nei neuroni che sopravvivono nei cervelli dei pazienti affetti dalla malattia. Il componente principale dei corpi di Lewy è una forma di alfa-sinucleina aggregata, fibrillare e ricca di foglietti beta. Il processo di aggregazione di alfa-sinucleina è stato ampiamente studiato negli anni passati: la proteina è non strutturata nella sua forma nativa, ma in condizioni patologiche tende ad aggregare formando specie oligomeriche. Questi oligomeri costituiscono un insieme etereogeneo e transiente e si convertono rapidamente in fibrille amiloidi quando raggiungono una concentrazione critica. Le fibrille amiloidi di alfa-sinucleina si depositano poi nei corpi di Lewy assieme ad altre proteine e lipidi. L’aggregazione di alfa-sinucleina è stata principalmente studiata in vitro, anche se più recentemente maggiori sforzi sono stati effettuati per caratterizzare il processo in modelli cellulari ed animali, per identificare non soltanto i diversi prodotti dell’aggregazione, ma anche i meccanismi tossici ad essi associati, che causano la morte dei neuroni nei pazienti affetti dalla malattia di Parkinson. In questa tesi due questioni principali sono state affrontate: lo studio dell’aggregazione di alfa-sinucleina in cellule utilizzando metodi non convenzionali di microscopia in fluorescenza e la caratterizzazione degli effetti di una famiglia di proteine chaperoniche, le 14-3-3, sul processo di aggregazione. Nella prima parte, due modelli cellulari per lo studio dell’aggregazione di alfa-sinucleina sono stati approntati e caratterizzati: il primo viene ottenuto sovraesprimento soltanto alfa-sinucleina e ha permesso la caratterizzazione di un ensemble di oligomeri eterogenei in cellule vive (circa 6±4 monomeri per oligomero) utilizzando un nuovo metodo di microscopia in fluorescenza chiamato Number and Brightness analysis. Queste specie oligomeriche inducono l’attivazione del sistema autofagico-lisosomiale e la frammentazione dei mitocondri in questo modello cellulare. Il secondo modello cellulare fornisce un metodo per lo studio delle fibrille di alfa-sinucleina e di aggregati più grandi in un ambiente di rilevanza fisiologica: alfa-sinucleina è stata sovrespressa in cellule e l’aggregazione è stata promossa introducendo nel citoplasma delle cellule frammenti di fibrille ottenute da alfa-sinucleina ricombinante, detti seeds. In entrambi i casi la sovraespressione e l’aggregazione di alfa-sinucleina hanno causato morte cellulare, in buon accordo con quello che è stato riportato in precedenza da altri gruppi di ricerca. La caratterizzazione dell’aggregazione di alfa-sinucleina in cellule è continuata osservando la variazione nel metabolismo cellulare, potenzialmente indotta da danni ai mitocondri. Queste variazione sono state quantificate misurando le proprietà della fluorescenza del NADH nei due modelli, rispetto al controllo. Questi risultati hanno mostrato che in cellule che presentano oligomeri o aggregati di alfa-sinucleina, il tempo di vita della fluorescenza del NADH e il suo spettro di emissione cambiano. Quindi, queste misure potrebbero essere ottimizzare per rilevare la presenza di aggregati di alfa-sinucleina in cellule e in vivo, utilizzando un metodo di indagine non invasivo e dye-free. La seconda parte della tesi riguarda l’abilità delle proteine chaperoniche 14-3-3 di interagire con alfa-sinucleina e di interferire con il suo processo di aggregazione, riducendone la tossicità in cellule. Tra le sette isoforme della famiglia di 14-3-3, la 14-3-3 eta può revertire il processo di fibrillazione di alfa-sinucleina in vitro, portando alla formazione di oggetti curvi invece che di fibrille canoniche. Questi oggetti curvi hanno diametri e curvature che dipendono dalla quantità di 14-3-3 eta nel saggio di aggregazione: inoltre, molecole di 14-3-3 eta sono state trovate in questi aggregati, suggerendo la formazione di un complesso stabile costituito dalle due proteine. Quanto la quantità di alfa-sinucleina è troppo grande o i seeds vengono utilizzati per promuovere il processo di aggregazione in vitro, la 14-3-3 eta non è più in grado di interferire con il processo di aggregazione di alfa-sinucleina e viene sequestrata nelle fibrille. Nei modelli cellulari, la sovraespressione di 14-3-3 eta riduce la tossicità indotta da alfa-sinucleina quando quest’ultima è soltato sovraespressa e oligomerizza, ma non quando l’aggregazione in cellule viene promossa dai seeds. È stato mostrato, utilizzando tecniche di image correlation spectroscopy (cross raster image correlation spectroscopy e cross Number and Brightness analysis) che la 14-3-3 eta sovraespressa può interagire con alfa-sinucleina sovraespressa, principalmente alla membrana plasmatica. Inoltre, la 14-3-3 eta viene sequestrata negli aggregati quando il processo di aggregazione di alfa-sinucleina è indotto dai seeds, evidenziando un altro possibile meccanismo di tossicità dovuto all’aggregazione. Tutti i risultati ottenuti in cellule sono in buon accordo con i risultati ottenuti in vitro e precedentemente riportati; questo rafforza ulteriormente l’idea che le proteine 14-3-3 e in particolare l’isoforma eta siano particolarmente interessanti nel contesto dello studio dell’aggregazione di alfa-sinucleina e che potrebbero essere utilizzare per interferire con il processo di aggregazione e ridurne gli effetti tossici.

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

The project of my PhD Thesis focuses on the general problem of the protein folding and misfolding in line with the research conducted in the laboratory of Protein Chemistry at CRIBI, where the work was mainly conducted. My research activity can be divided in two parts. In the first year of the PhD course I studied the effect of pH in protein fibrillogenesis using a peptide model. During the second and the third years, my research was focused into the molecular interaction between alpha-synuclein and fatty acids and its implications in alpha-synuclein aggregation. Thus, this PhD Thesis is composed of a minor part concerning the analysis of the aggregative properties of the peptide model apoMb1-29 (Chapter 1 and 2) and of a major part dealing with the characterization of the interaction of alpha-synuclein and fatty acids (Chapter 3 and 4). Several human diseases, defined also misfolding disease, result from the failure of protein folding of the involved proteins. An increasing number of human diseases, such as Alzheimer’s and Parkinson’s diseases (PD), have been linked to protein aggregation and the aberrant accumulation of protein deposits in different tissues and organs. These pathological deposits are characterized by the presence of highly organized fibrillar aggregates called amyloid fibrils. Amyloid is a non-covalent polymer of extended, intermolecularly hydrogen bonded betha-sheets that laterally self-assemble to yield twisted fibers. Since amyloid fibrils are formed from disease-associated as well as from disease unrelated proteins and peptides under appropriate conditions, there is the belief that the ability to form fibrils is a generic property of the polypeptide chain (Chiti and Dobson, 2006). However, the propensity to aggregate and the stability of the mature fibrils depends on the amino acid sequence, so intrinsic determinants, such as net charge, hydrophobicity, the presence of aromatic residues and betha-sheet propensity, have important roles in amyloidogenicity of polypeptides (Pawar et al., 2005). In order to investigate the role of the net charge in the aggregation process of unfolded proteins and to analyze the importance of electrostatic interaction in the stability of the resulting fibrils, the aggregation properties of a peptide model derived from the N-terminal region of apomyoglobin were analyzed under different pH conditions. The N-terminal fragment 1-29 of horse heart apomyoglobin (apoMb1-29) is highly prone to form amyloid-like fibrils at low pH. Fibrillogenesis at pH 2.0 occurs following a nucleation-dependent growth mechanism, as evidenced by the thioflavin T (ThT) assay. Transmission electron microscopy (TEM) confirms the presence of regular amyloid-like fibrils and far-UV circular dichroism (CD) spectra indicate the acquisition of a high content of betha-sheet structure. Using peptides deriving from the proteolysis of apoMb1–29, we identified the region 7-16 as the most amyloidogenic, indeed it contains in terms of hydrophobicity, betha-sheet propensity and low net charge, all the determinants that favor the aggregation. In conclusion, the modulation of the net charge of apoMb1-29 and its sub-fragments by change of pH is of utmost importance for fibril formation. Moreover, we demonstrated that the electrostatic interaction, in apoMb1-29 system, is the force that primarily stabilizes the betha-sheet structure of the mature fibrils. Indeed, ThT assay, TEM and CD highlight fast and complete disaggregation of the fibrils, if the pH of a suspension of mature fibrils is increased to neutral values. In the second part of my PhD project, I investigated the molecular details that regulate the interaction between alpha-synuclein (alpha-syn) and fatty acids (FAs), analyzing the conformational features of the protein bound to FAs and the physical state of the lipids. Moreover, the aggregation process FA-mediated was analyzed in order provides insights into the implication of lipids in amyloid formation in vivo. Human alpha-syn is a 140 amino acid natively unfolded protein of still unknown function. It is highly expressed in the central nervous system and enriched in the presynaptic nerve terminals. alpha-Syn is characterized by 7 repetitive amino acid sequences (KTKEGV) in the N-terminal portion, by a central hydrophobic region (non-amyloid component, NAC) and by acidic stretches in the C-terminal tail. Mutations or overexpression of the human alpha-syn gene cause early-onset autosomal dominant Parkinson’s disease (PD). alpha-Syn is the major component of Lewy bodies, the cytoplasmic proteinaceous aggregates pathognomonic for PD (Spillantini et al., 1998). The mechanism by which an abnormality in structure or expression of alpha-syn causes PD has not been elucidated. Despite the evidence for a key role of alpha-syn in the onset of PD, there is very little information about its physiological function in the brain. Among several hypotheses, the role of alpha-syn is also associated to FAs. alpha-Syn seems to interact with unsatured and polyunsatured fatty acids (PUFAs), but it is not known if this interaction involves free FA molecules (Sharon et al., 2001), or aggregate states of FAs (micelles, vesicles, oil droplets) (Broersen et al., 2006; Lücke et al., 2006). Furthermore, this interaction promotes the oligomerization of alpha-syn. alpha-Syn forms multimers in vitro upon exposure to vesicles containing certain PUFA acyl groups and this process occurs at physiological concentration (Perrin et al., 2001). Moreover, since exposure of neuronal cell lines to PUFA increases alpha-syn oligomer levels, the in vivo interaction of alpha-syn with PUFAs seems to promote the formation of soluble oligomers that precede the aggregates associated with neurodegeneration (Sharon et al., 2003). First, a systematic study on the conformational transitions of alpha-syn in the presence of several fatty acids was conducted. Since the number of unsaturations and the length of the acyl chain have been shown to deeply affect the aggregate state of fatty acid (monomer, micelle, vesicle and oil droplet) and consequently, the interaction with the protein, the analysis was conducted using several fatty acids: palmitic acid (saturated), oleic acid (unsaturated), and docosahexaenoic acid (DHA, polyunsaturated). In particular, the last one is an essential omega-3 fatty acid, abundant in brain. DHA levels have been shown to be elevated in those brains areas containing alpha-syn inclusions in PD patients (Sharon et al., 2003). The FAs effects on alpha-syn structure were analyzed by far-UV circular dichroism and by proteolytic mapping. The protein is unfolded in the absence of FAs or in the presence of palmitic acid. Instead, upon binding to oleic acid (OA) and DHA, alpha-syn acquires alpha-helical conformation in a simple two-state transition. In the presence of DHA, alpha-syn is quite resistant to proteolysis by proteinase K and trypsin. We reported that the segment 70-90 in the NAC region is more susceptible to proteolytic attack than the N-terminal region. Probably, This region is flexible and sufficiently protruded to be protease-sensitive even if the analysis of CD spectra in the far-UV demonstrates that this region has an alpha-helix conformation and the NMR experiment indicates that only the C-terminal ~ 40 residues continue to be unfolded and mobile in the presence of DHA. Furthermore, we observed that alpha-syn strongly affects the self-association process of DHA. The physical state of the lipid in the presence of the protein was analyzed by turbidity measurements, dynamic light scattering (DLS), pyrene fluorescence analysis and transmission electron microscopy (TEM). At pH 7.4, DHA assembles in oil droplets with a large size distribution (Namani et al., 2007). alpha-Syn disrupts these lipid aggregates, stabilizing a new product of DHA self-assembly. These species are formed at lower concentrations range and they have a regular shape, a smaller diameter and a reduced hydrophobic volume. Truncated forms of alpha-syn corresponding to different parts of its polypeptide chain (syn1-99, syn1-52, syn57-102, and syn108-140) were also used to extend the knowledge on the role of different protein regions in the interaction with the lipid. CD data suggest that there is an important role of the repeats in the alpha-helix transition and thereby in the interaction with DHA. The C-terminal region, at variance, seems to modulate the portion of alpha-syn buried into the lipid compartment. Moreover, with the exception of syn 108-140, all the polypeptides affect the self-assembly process of DHA. We can hypothesize that the N-terminal region of alpha-syn has a crucial role even in the regulation of DHA aggregation process. Finally, a general consideration concerns the ability of DHA and probably of other long chain PUFAs to induce oligomerization and fibrillation of alpha-syn (Perrin et al., 2001; Sharon et al., 2003; Broersen et al., 2006). The molecular effect of DHA on aggregation process of alpha-syn was analyzed by CD, native gel electrophoresis, Thioflavin T assay and TEM observation. The presence of DHA, in a molar ratio [DHA]/[alpha-syn] of 10, promotes aggregation and fibrils formation of alpha-syn. On the contrary, in the presence of saturating conditions of DHA, only oligomeric species are formed. DHA exerts a direct effect on protein structure, stabilizing an amyloidogenic conformation and generates an environment that can promote protein aggregation. Sharon R., Goldberg M.S., Bar-Josef I., Betensky R.A., Shen J., Selkoe D.J. (2001). Alpha-Synuclein occurs in lipid-rich high molecular weight complexes, binds fatty acids, and shows homology to the fatty acid-binding proteins. Proc. Natl. Acad. Sci. U S A. 98(16), 9110?9115. Broersen K., van den Brink D., Fraser G., Goedert M., Davletov B. (2006). Alpha-synuclein adopts an alpha-helical conformation in the presence of polyunsaturated fatty acids to hinder micelle formation. Biochem. 45(51), 15610?15616. Chiti, F. and Dobson, C. M. (2006). Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem., 75, 333?366. Lücke C., Gantz D. L., Klimtchuk E., Hamilton J. A. (2006). Interactions between fatty acids and alpha-synuclein. J. Lipid Res. 47, 1714?1724. Namani T., Ishikawa T., Morigaki K., Walde P. (2007). Vesicles from docosahexaenoic acid. Colloids and Surfaces B: Biointerfaces. 54, 118?123. Pawar, A. P., DuBay, K. F., Zurdo, J., Chiti, F., Vendruscolo, M. & Dobson, C. M. (2005). Prediction of “aggregation-prone” and “aggregation-susceptible” regions in proteins associated with neurodegenerative disease. J. Mol. Biol. 350, 379?392. Perrin R.J., Woods W.S., Clayton D.F., George J.M. (2001). Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins. J. Biol. Chem. 276(45), 41958?41962. Sharon R., Bar-Joseph I., Frosch M.P., Walsh D.M., Hamilton J.A., Selkoe D.J. (2003). The formation of highly soluble oligomers of alpha-synuclein is regulated by fatty acids and enhanced in Parkinson's disease. Neuron. 37(4), 583?595. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M. & Goedert, M. (1998). alpha-Synuclein in filamentous inclusions of Lewy bodies from Parkinson's disease and dementia with Lewy bodies. Proc. Natl. Acad. Sci. U S A, 95, 6469–6473.
Il progetto della mia Tesi di dottorato riguarda il problema del folding di proteine ed il loro misfolding, in linea con la ricerca condotta nel laboratorio di Chimica delle Proteine dove è stato principalmente svolto lo studio. La ricerca svolta può essere divisa in due parti. Durante il primo anno di dottorato è stato studiato l’effetto del pH nella fibrillogenesi di proteine, mediante l’analisi delle caratteristiche di un peptide modello. Nel secondo e terzo anno di dottorato, è stata analizzato il complesso formato da alpha-sinucleina umana ed acidi grassi e le implicazioni di questa interazione nel processo di aggregazione della proteina. Di conseguenza, la Tesi è composta da una prima parte riguardante lo studio delle proprietà di aggregazione del peptide apoMb1-29 (Capitolo 1, 2) e di una seconda parte dedicata alla caratterizzazione dell’interazione di alpha-sinucleina con acidi grassi (Capitolo 3, 4). Molte malattie umane, definite anche misfolding diseases, derivano da una non corretta strutturazione delle proteine coinvolte. Un numero sempre maggiore di malattie, come il morbo di Alzheimer e di Parkinson, è correlato al fenomeno dell’aggregazione proteica e all’accumulo anomalo di depositi proteici in diversi tessuti e organi. Questi depositi patologici sono formati da aggregati proteici fibrillari, chiamati fibrille amiloidi. L’amiloide è un polimero proteico non-covalente, stabilizzato da struttura di tipo beta, in cui i diversi betha-strands sono lateralmente associati e formano aggregati fibrillari. Poiché anche proteine e peptidi non direttamente coinvolti in patologie sono in grado di formare fibrille amiloidi in appropriate condizioni, si ritiene che la capacità di formare fibrille sia una proprietà generica delle backbone polipeptidico (Chiti and Dobson, 2006). Comunque, la tendenza ad aggregare e la stabilità delle fibrille dipende dalla sequenza aminoacidica, quindi determinanti intrinseci, come la carica netta, l’idrofobicità, la presenza di residui aromatici e la propensione a formare struttura beta, hanno un ruolo determinante nell’amiloidogenicità di una catena polipeptidica (Pawar et al., 2005). Per comprendere l’importanza della carica netta di una proteina nel suo processo di aggregazione e per analizzare gli effetti dell’interazione elettrostatica nella stabilità delle risultanti fibrille, le proprietà di aggregazione di un peptide, corrispondente al frammento 1-29 di apomioglobina da cuore di cavallo (apoMb1-29), sono state studiate in differenti condizioni di pH. Questo peptide forma velocemente fibrille amiloidi a pH acidi. Il processo a pH 2.0 segue un meccanismo di crescita nucleazione-dipendente, come determinato dall’analisi fluorimetrica mediante Tioflavina T (ThT). Osservazioni mediante microscopia elettronica (TEM) confermano la presenza di fibrille e misure di dicroismo circolare (CD) indicano l’acquisizione di un alto contenuto di struttura secondaria di tipo beta. Mediante l’uso di peptidi derivanti dalla proteolisi di apoMb1-29, è stata poi identificata la regione 7-16 come la più amiloidogenica, infatti, ha un alto grado di idrofobicità, propensione a formare beta-sheet e bassa carica netta. In conclusione, la modulazione della carica netta dei peptidi analizzati, derivante da un cambiamento del pH, è il fattore che primariamente regola formazione di aggregati fibrillari. Inoltre, è stato dimostrato che interazioni di tipo elettrostatico hanno un ruolo determinante anche nel stabilizzare la struttura beta di fibrille mature. Infatti, ThT, TEM e CD hanno evidenziato una veloce e completa disaggregazione delle fibrille, se il pH della sospensione viene portato a valori più basici. Nella seconda parte del mio progetto di dottorato, ho studiato i dettagli molecolari che regolano l’interazione tra alpha-sinucleina (alpha-syn) e acidi grassi, analizzando sia le caratteristiche conformazionali della proteina acquisite in presenza dell’acido grasso, sia lo stato fisico dello stesso lipide. Inoltre, è stato studiato il processo di aggregazione di alpha-syn mediato da acidi grassi, allo scopo di comprendere l’implicazione dei lipidi nella formazione amiloide in vivo. ?-Sinucleina è una proteina solubile di 140 aminoacidi, natively unfolded con funzione sconosciuta. Essa è altamente espressa nel sistema nervoso centrale ed è abbondante nei terminali presinaptici dei neuroni. Questa proteina è caratterizzata dalla presenza di sette ripetizioni imperfette di sequenza aminoacidica (KTKEGV) nella regione N-terminale, da una regione idrofobica centrale (NAC, non-amyloid component) e da una coda C-terminale che presenta numerosi residui acidi. La sovraespressione di ?-syn e mutazioni nel suo gene sono associati a forme precoci della sindrome di Parkinson. Inoltre, alpha-syn è il componente principale dei corpi di Lewy, accumuli citoplasmatici caratteristici del morbo di Parkinson (Spillantini et al., 1998). Il meccanismo con cui un cambiamento nella struttura e nell’espressione della proteina possa portare allo sviluppo della malattia non è ancora stato chiarito. Nonostante l’evidenza di un ruolo chiave nella patogenesi, ci sono ancora poche informazioni sulla funzione fisiologica di alpha-syn a livello neuronale. Tra le varie ipotesi, la funzione di alpha-syn è stata associata anche ad acidi grassi. alpha-Syn sembra essere in grado di interagire con acidi grassi insaturi e polinsaturi, ma non è ancora chiaro se l’interazione coinvolga molecole libere (Sharon et al., 2001), o stati aggregati (micelle, vescicole, oil droplets) di acidi grassi (Broersen et al., 2006; Lücke et al., 2006). Questa interazione modula anche l’oligomerizzazione della proteina. Infatti, studi in vitro hanno evidenziato come alpha-Syn formi multimeri in seguito all’esposizione a vescicole formate da lipidi contenenti PUFA (Perrin et al., 2001). Inoltre, in linee cellulari neuronali trattate con PUFA è stato descritto un aumento della formazione di oligomeri di alpha-syn. Queste strutture potrebbero precedere la formazione di aggregati associati alla neurodegenerazione (Sharon et al., 2003). In questo lavoro di tesi è stato effettuato in primo luogo uno studio sistematico sulle transizioni conformazionali di alpha-syn in presenza di diversi acidi grassi. Dato che il numero di insaturazioni e la lunghezza della catena acilica hanno un importante effetto sullo stato aggregativo dell’acido grasso (monomero, micella, vescicola o oil droplet) e di conseguenza anche nell’interazione con la proteina, l’analisi è stata condotta usando acidi grassi con diverse caratteristiche: acido palmitico (saturo), acido oleico (monoinsaturo) e acido docosaesaenoico (DHA, polinsaturo). Quest’ultimo è un acido grasso omega-3 abbondante a livello delle membrane neuronali. E’ stato osservato che in aree del cervello di pazienti affetti da morbo di Parkinson contenenti inclusioni di alpha-syn, si registra un aumento nel livello di DHA. Gli effetti degli acidi grassi sulla struttura di alpha-syn sono stati analizzati mediante CD e mapping proteolitico. La proteina è unfolded in assenza degli acidi grassi e in presenza di acido palmitico, mentre in seguito al legame con acido oleico e DHA, acquisisce una conformazione alpha-elicoidale mediante una semplice transizione a due stadi. In presenza di DHA, alpha-syn è abbastanza resistente alla proteolisi con proteinasi K e tripsina e il segmento 70-90 contenuto nella regione NAC è maggiormente suscettibile all’attacco proteolitico rispetto alla regione N-terminale. Probabilmente, questo segmento è flessibile ed sufficientemente esposto all’azione proteolitica, nonostante l’analisi CD dimostri la presenza di struttura alpha-elica e gli esperimenti NMR indichino che solo 40 residui del C-terminale risultano essere destrutturati e mobili in presenza di DHA. Successivamente, abbiamo osservato che alpha-syn altera il processo di auto-associazione di DHA. Lo stato fisico del lipide in presenza di alpha-syn è stato analizzato mediante misure di torbidità, dynamic light scattering (DLS), TEM e studi di fluorescenza utilizzando il pirene come sonda. DHA forma oil droplets polidisperse a pH neutro (Namani et al., 2007). alpha-Syn disgrega questi aggregati lipidici, favorendo una diversa forma di auto associazione di DHA. In presenza di alpha-syn sono necessarie concentrazioni minori di acido grasso per ottenere questa specie, caratterizzata da forma più regolare, diametro inferiore e volume idrofobico ridotto. Forme tronche di alpha-syn corrispondenti a diverse parti della catena polipeptidica (syn1-99, syn1-52, syn57-102, syn108-140) sono state utilizzate per ulteriori studi sul ruolo che ciascuna regione ha nell’interazione con il lipide. Analisi CD evidenziano come le sequenze ripetute svolgano un’importante funzione nella transizione ad alpha-elica e, di conseguenza, nell’interazione con DHA. Invece, la regione C-terminale sembra modulare la porzione di proteina che si colloca nel compartimento lipidico. Questi peptidi sono stati utilizzati anche nello studio delle proprietà aggregative di DHA. Ad eccezione di syn108-140, tutti gli altri peptidi alterano il processo di auto-associazione di DHA. Possiamo, quindi, ipotizzare che la regione N-terminale svolge un ruolo cruciale anche nel regolare il processo aggregativo di DHA. Infine, in questo lavoro di tesi si discute l’abilità del DHA, e probabilmente di altri acidi grassi polinsaturi, di indurre la formazione di oligomeri e fibrille di alpha-syn (Perrin et al., 2001; Sharon et al., 2003; Broersen et al., 2006). Gli effetti molecolari di DHA sull’aggregazione di alpha-syn sono stati analizzati mediante CD, elettroforesi su gel nativo, ThT e TEM. La presenza di DHA in un rapporto molare [DHA]/[alpha-syn] di 10, promuove l’aggregazione e la formazione di fibrille della proteina. Al contrario, condizioni saturanti di DHA inducono la formazione di sole specie oligomeriche. DHA esercita un effetto diretto sulla struttura proteica, stabilizzandone una conformazione amiloidogenica, e crea un ambiente che promuove l’aggregazione proteica.

Els trastorns neurodegeneratius crònics, condicions mèdiques que afecten a la població principalment a la seva darrera etapa de vida, representen un problema molt important a la societat moderna. Per això, trobar nous mètodes de diagnòstic i teràpies per tractar aquestes patologies representa un objectiu que cada vegada es presenta com més urgent. Les malalties neurodegeneratives estan caracteritzats pel malplegament proteic intra i extracel•lular, que deriva en la formació d’agregats ordenats responsables de l’inici d’aquestes patologies. Per altra banda, l’agregació representa la major limitació en la producció d’agents terapèutics de caràcter proteic. Per desvetllar les causes que es troben darrera de la formació d’aquests dipòsits insolubles, els mecanismes pels quals provoquen toxicitat cel•lular i com l’evolució enfronta aquest risc, hem utilitzat un enfoc multidisciplinar per tal d’estudiar els determinants de les reaccions d’agregació, utilitzant diferents models proteics com el pèptid β-amiloide i l’α-sinucleïna. La recerca que es presenta en aquesta tesi persegueix entendre els determinants de l’agregació proteica i la toxicitat que s’hi associa, tant en l’entorn cel•lular com en condicions in vitro, així com investigar com la pressió selectiva ha modelat els proteomes al llarg de l’evolució per tal d’evitar l’agregació. Implementant una nova eina bioinformàtica basada en l’estructura tridimensional de les proteïnes, hem identificat els principals determinants estructurals de l’agregació proteica utilitzant bacteris com a organisme model. Es coneix molt poc sobre els determinants estructurals que condueixen l’agregació d’una proteïna cap a una via determinada i com aquests resulten en diverses estructures macromoleculars agregades que exhibeixen diferent toxicitat. En aquest treball, abordem aquest problema usant l’α-sinucleïna, proteïna intrínsecament desordenada associada a la malaltia de Parkinson. Finalment, utilitzant una altra proteïna intrínsecament desordenada, el pèptid β-amiloide i mutants d’aquest, hem identificat les espècies conformacionals responsables del dany oxidatiu cel•lular causat per l’agregació d’aquest pèptid relacionat amb l’Alzheimer, utilitzant llevat com a sistema model. De manera global, el treball d’aquesta tesi pretén entendre aspectes fonamentals del procés d’agregació proteic, tant en organismes procariòtics com en eucariòtics, il•lustrant cóm la la integració de diferent disciplines pot millorar el nostre coneixement sobre l’impacte de l’agregació de proteïnes en la salut i la malaltia.
Chronic neurodegenerative disorders, the medical conditions that strike primarily mid- to late-life population, represent a major issue of modern society. Therefore, finding new diagnostic and therapeutic approaches to treat these disorders is a goal of increasing urgency. The neurodegenerative disorders are characterised by intra/extracellular protein misfolding, resulting in the formation of ordered aggregates that are responsible for the onset of these diseases. On the other hand, aggregation represents a major limitation in the industrial production of proteinaceous therapeutic agents. To elucidate the causes behind the formation of these insoluble deposits, the mechanisms by which they mediate cellular toxicity, and how evolution confronts this risk, we employed a multidisciplinary approach to study the determinants of aggregation reactions, using different protein models such as, amyloid β-peptide and α-synuclein. The research presented in this thesis seeks to understand the determinants of protein aggregation and its associated toxicity, in both the cellular environment and in vitro conditions, as well as to investigate how the selective pressure that acts to to avoid the aggregation has shaped the cellular proteomes along the evolution. Implementing a novel structure-based bioinformatic tool, we identify the structural determinants of protein aggregation using bacteria as a model organism. Little is known on the structural determinants that drive the aggregation of a protein to a particular pathway, resulting in diverse aggregated macromolecular structures displaying different toxicity. Here, we address this issue using the Parkinson’s disease-associated intrinsically disordered protein, α-synuclein. Finally, using another intrinsically disordered protein, the amyloid β-peptide and mutants thereof, we identify the conformational species responsible for the cellular oxidative damage caused by the aggregation of this Alzheimer’s linked peptide, employing yeast as a model system. Overall, the work in this thesis attempts to understand fundamental aspects of protein aggregation processes, in both prokaryotic and eukaryotic organisms, highlighting how the interplay between different disciplines might improve our understanding on the impact of protein aggregation in health and disease.

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

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

L'atrophie multisystematisée (AMS) est une maladie neurodégénérative rare et à évolution rapide qui affecte de nombreuses régions du système nerveux central, y compris les systèmes olivopontocérébelleux et striatonigral ainsi que divers noyaux autonomes du tronc cérébral. La caractéristique pathologique de l'AMS est la présence d'agrégats oligodendrogliaux appelés inclusions cytoplasmiques gliales dont le composant principal est la protéine a-synuclein. Le processus neurodégénératif conduit à une dysautonomie, ainsi qu'à un degré variable de syndromes parkinsoniens et cérébelleux. Il n'existe actuellement aucun traitement pour ralentir l'évolution de la maladie. Ce travail de thèse a porté, d'une part, sur des approches précliniques visant à réduire l'accumulation de l'a-synuclein dans un modèle de souris transgénique de l'AMS, et d'autre part, sur une analyse anatomo-pathologique chez des patients présentant une forme lentement progressive de la maladie. Tout au long de ces trois années de travail, nous avons évalué différents candidats thérapeutiques chez la souris transgénique de l'AMS. La rapamycine, un médicament connu pour stimuler l'autophagie et la clairance des protéines, n'a montré qu'un effet neuroprotecteur partiel contre la perte neurale dans notre modèle. Le nilotinib, un médicament qui avait démontré des propriétés neuroprotectrices dans un modèle rongeur de la maladie de Parkinson, n'a pas eu d'effet sur l'accumulation de l'a-synuclein et la neurodégénérescence. Enfin, nous avons évalué la combinaison de deux médicaments (anle138b et belnacasan) qui ont déjà démontré leur capacité à réduire l'agrégation de l'a-synucléine et à protéger les neurones de la dégénérescence, pour déterminer si elles ont des effets synergiques
Multiple system atrophy (MSA) is a rare, rapidly progressive neurodegenerative disease that affects numerous regions of the central nervous system, including the olivopontocerebellar and striatonigral systems as well as various autonomous nuclei of the brainstem. The pathological hallmark of MSA is the presence of oligodendroglial aggregates called glial cytoplasmic inclusions (GCI) whose main component is the protein ¦Á-synuclein. The neurodegenerative process leads to severe impairment of autonomic dysfunction, together with a varying degree of parkinsonian and cerebellar syndromes. There is currently no disease modifying therapy available. This PhD work focused, on the one hand, on preclinical approaches aiming to reduce the accumulation of ¦Á-synuclein in an animal model of MSA, and on the other hand, on a neuropathological analysis in patients with a slowly progressive subtype of the disease. Throughout the three years of work, we have assessed different therapeutic candidates in an animal model of MSA. Rapamycin, a drug known to enhance autophagy and protein clearance, showed only a partial neuroprotective effect against neural loss in our model. Nilotinib, a drug that had shown neuroprotective properties in a Parkinson¡¯s disease animal model, failed to modify the disease course in our study. Finally, we evaluated the combinations of two drugs that have already proven to reduce a-synuclein aggregation and protect neurons from degeneration, to assess whether they have synergistic properties.Keywords: Synuclein, Multiple system atrophy, glial cytoplasmic inclusions, post-mortem human brain study, rodent, translational approach, c-terminal truncation, phosphorylation, autophagy, protein aggregation

The research work performed during the doctorate focused on the computational aspects of the kinetics of aggregation and interaction of amyloidogenic proteins involved in neurological disorders. Incorrectly folded proteins may lose their colloidal stability, resulting in the formation of soluble oligomers and insoluble amyloidogenic aggregates. Amyloid fibrils are protein aggregates characterized by a filamentous β-sheet-rich structure. Although specific amyloidogenic proteins, such as α-synuclein (α-syn), β-amyloid (Aβ), huntingtin, prion protein (PrP), etc., are known to be involved in neurodegenerative diseases, the current understanding of fibril formation mechanisms implies that at certain (sometimes non-physiological) conditions almost every protein may form fibrillary structures. In the human organism, although there are few evidences of functional and physiological amyloids, these structures generally lead to amyloidosis by forming insoluble plaques, which accumulate in tissues and organs, leading to disruption of their normal functions. This doctorate thesis dealt with different problems concerning the study of neurodegenerative diseases, particularly focusing on kinetics of aggregation, new diagnostic strategies, drug discovery and drug screening methodologies

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.
Source: Dissertation Abstracts International, Volume: 69-11, Section: B, page: 6601. Advisers: David F. Clayton; Julia M. George. Includes bibliographical references (leaves 157-199) Available on microfilm from Pro Quest Information and Learning.

α-Synuclein (AS) is a small intrinsically disordered protein expressed in neurons and abundantly present in synapses where it is involved in regulation of synaptic vesicle-mediated protein trafficking. Misfolding of AS into amyloid fibrils is a key process in progression of Parkinson's disease (PD), the second most common neurodegenerative disorder which has no cure to date. Inhibition of AS aggregation and blocking of cell-to-cell spreading of AS fibrils is a promising strategy for PD treatment. However, rational design of inhibitors of this type remains complicated due to the lack of thorough knowledge about the mechanisms of aggregation. Therefore, the aim of this thesis was to gain deeper knowledge about AS aggregation and to apply it for developing inhibitors of AS fibrillization. In my work on the mechanisms of AS aggregation, I first determined that the concentration of AS that enables the fibril growth is an order of magnitude lower than the concentration of AS required for initial fibril formation from monomers. I explored fibril disaggregation at AS concentrations below its Kd value, and characterized AS aggregation at low micromolar concentrations. I then investigated how different modifications of AS C-terminus (namely, extensions of various sizes and charges) affect fibril growth and...

My Ph.D. research project, entitled “Expression and characterization of human proteins involved in neurodegenerative diseases”, was focused on the application of molecular biology and proteomics methodologies to prepare samples of proteins involved in neurodegenerative diseases. The target proteins were α-synuclein (α-syn) and the amyloid-beta peptides (Aβ), involved in the pathogenesis of Parkinson’s disease (PD) and Alzheimer’s disease (AD), respectively. The aim of my research activity was the optimization of the expression and purification of the neurodegeneration-associated proteins to carry out the following projects:  “NMR analysis of the aggregation kinetics of Aβ1-40”, to reveal aggregation mechanisms of Aβ1-40 and to develop a kinetic model describing the formation of oligomeric and fibrillary species.  “NMR analysis of the assembly of Aβ42: Aβ40 mixed fibrils”. This project is in the frame of an integrative study aimed to characterize the structure of the mixed fibrils (containing Aβ1-42 and Aβ1-40 peptides) at atomic detail.  “Development of a protein aggregation assays for the diagnosis of synucleinopathies” This project was focused on the development of a protocol tool for the diagnosis PD, dementia with Lewy bodies (DLB) and multiple system atrophy (MSA) based on α-synuclein aggregation assays (SAA-seeding aggregation assays) starting from aliquots of Cerebrospinal fluid (CSF).  Study of the interaction between alpha-synuclein and human biofluids components” This project concerned the study of the interaction of α-synuclein with lipoproteins, proteins and other constituents of CSF and plasma.