Добірка наукової літератури з теми "Alpha-Synuclein, Prion Protein, Aggregation"

Among nutraceuticals, phytochemical-rich compounds represent a source of naturally-derived bioactive principles, which are extensively studied for potential beneficial effects in a variety of disorders ranging from cardiovascular and metabolic diseases to cancer and neurodegeneration. In the brain, phytochemicals produce a number of biological effects such as modulation of neurotransmitter activity, growth factor induction, antioxidant and anti-inflammatory activity, stem cell modulation/neurogenesis, regulation of mitochondrial homeostasis, and counteracting protein aggregation through modulation of protein-folding chaperones and the cell clearing systems autophagy and proteasome. In particular, the ability of phytochemicals in restoring proteostasis through autophagy induction took center stage in recent research on neurodegenerative disorders such as Parkinson’s disease (PD). Indeed, autophagy dysfunctions and α-syn aggregation represent two interdependent downstream biochemical events, which concur in the parkinsonian brain, and which are targeted by phytochemicals administration. Therefore, in the present review we discuss evidence about the autophagy-based neuroprotective effects of specific phytochemical-rich plants in experimental parkinsonism, with a special focus on their ability to counteract alpha-synuclein aggregation and toxicity. Although further studies are needed to confirm the autophagy-based effects of some phytochemicals in parkinsonism, the evidence discussed here suggests that rescuing autophagy through natural compounds may play a role in preserving dopamine (DA) neuron integrity by counteracting the aggregation, toxicity, and prion-like spreading of α-syn, which remains a hallmark of PD.

AbstractThe protein α-synuclein, a key player in Parkinson’s disease (PD) and other synucleinopathies, exists in different physiological conformations: cytosolic unfolded aggregation-prone monomers and helical aggregation-resistant multimers. It has been shown that familial PD-associated missense mutations within the α-synuclein gene destabilize the conformer equilibrium of physiologic α-synuclein in favor of unfolded monomers. Here, we characterized the relative levels of unfolded and helical forms of cytosolic α-synuclein in post-mortem human brain tissue and showed that the equilibrium of α-synuclein conformations is destabilized in sporadic PD and DLB patients. This disturbed equilibrium is decreased in a brain region-specific manner in patient samples pointing toward a possible “prion-like” propagation of the underlying pathology and forms distinct disease-specific patterns in the two different synucleinopathies. We are also able to show that a destabilization of multimers mechanistically leads to increased levels of insoluble, pathological α-synuclein, while pharmacological stabilization of multimers leads to a “prion-like” aggregation resistance. Together, our findings suggest that these disease-specific patterns of α-synuclein multimer destabilization in sporadic PD and DLB are caused by both regional neuronal vulnerability and “prion-like” aggregation transmission enabled by the destabilization of local endogenous α-synuclein protein.

Parkinson’s disease (PD) is a debilitating neurodegenerative disorder characterized by progressive motor decline and the aggregation of α-synuclein protein. Growing evidence suggests that α-synuclein aggregates may spread from neurons of the digestive tract to the central nervous system in a prion-like manner, yet the mechanisms of α-synuclein transmission and neurotoxicity remain poorly understood. Animal models that are amenable to high-throughput investigations are needed to facilitate the discovery of disease mechanisms. Here we describe the first Caenorhabditis elegans models in which feeding with α-synuclein preformed fibrils (PFFs) induces dopaminergic neurodegeneration, prion-like seeding of aggregation of human α-synuclein expressed in the host, and an associated motor decline. RNAi-mediated knockdown of the C. elegans syndecan sdn-1, or other enzymes involved in heparan sulfate proteoglycan synthesis, protected against PFF-induced α-synuclein aggregation, motor dysfunction, and dopamine neuron degeneration. This work offers new models by which to investigate gut-derived α-synuclein spreading and propagation of disease.

The protein alpha-synuclein (αS) self-assembles into small oligomeric species and subsequently into amyloid fibrils that accumulate and proliferate during the development of Parkinson’s disease. However, the quantitative characterization of the aggregation and spreading of αS remains challenging to achieve. Previously, we identified a conformational conversion step leading from the initially formed oligomers to more compact oligomers preceding fibril formation. Here, by a combination of single-molecule fluorescence measurements and kinetic analysis, we find that the reaction in solution involves two unimolecular structural conversion steps, from the disordered to more compact oligomers and then to fibrils, which can elongate by further monomer addition. We have obtained individual rate constants for these key microscopic steps by applying a global kinetic analysis to both the decrease in the concentration of monomeric protein molecules and the increase in oligomer concentrations over a 0.5–140-µM range of αS. The resulting explicit kinetic model of αS aggregation has been used to quantitatively explore seeding the reaction by either the compact oligomers or fibrils. Our predictions reveal that, although fibrils are more effective at seeding than oligomers, very high numbers of seeds of either type, of the order of 104, are required to achieve efficient seeding and bypass the slow generation of aggregates through primary nucleation. Complementary cellular experiments demonstrated that two orders of magnitude lower numbers of oligomers were sufficient to generate high levels of reactive oxygen species, suggesting that effective templated seeding is likely to require both the presence of template aggregates and conditions of cellular stress.

Earlier we showed that derivatives of hydroxycinnamic acids prevent amyloid transformation of alpha-synuclein and prion protein. The aim of this work was to determine the content of 3-hydroxycinnamic acid derivatives in coffee extracts and to evaluate their activity in relation to alpha-synuclein amyloid aggregation. Hydroxycinnamic acid derivatives were identified in aqueous and ethanol extracts of coffee beans by quantitative mass spectrometric analysis. Only 3,4-dimethoxycinnamic acid (13–53 μg/mL) was detected in significant amounts in the coffee extracts, while ferulic acid was present in trace amounts. In addition, 3-methoxy-4-acetamidoxycinnamic acid (0.4–0.8 μg/mL) was detected in the roasted coffee extracts. The half-maximum inhibitory concentrations of alpha-synuclein fibrillization reaction in the presence of coffee extracts, as well as inhibitory constants, were determined using thioflavin T assay. The inhibitory effect of black and green coffee extracts on alpha-synuclein fibrillization is dose-dependent, and in a pairwise comparison, the constants of half-maximal inhibition of fibrillization for green coffee extracts are comparable to or greater than those for black coffee. Thus, coffee extracts prevent pathological transformation of alpha-synuclein in vitro, probably due to the presence of 3,4-dimethoxycinnamic acid in them. Consequently, coffee drinks and coffee extracts can be used for the prevention of synucleinopathies including Parkinson’s disease.

Prions are proteins that adopt alternative conformations that become self-propagating; the PrPSc prion causes the rare human disorder Creutzfeldt–Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T–YFP) and TgM83+/− mice expressing α-synuclein (A53T). The TgM83+/− mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83+/+ mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83+/− mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T–YFP in cultured cells, whereas none of six Parkinson’s disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83+/+ mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.

Proteins aggregate in several slowly progressive neurodegenerative diseases called ‘proteinopathies’. Studies with cell cultures and transgenic mice overexpressing mutated proteins suggested that aggregates of one protein induced misfolding and aggregation of other proteins as well – a possible common mechanism for some neurodegenerative diseases. However, most proteinopathies are ‘sporadic’, without gene mutation or overexpression. Thus, proteinopathies in WT animals genetically close to humans might be informative. Squirrel monkeys infected with the classical bovine spongiform encephalopathy agent developed an encephalopathy resembling variant Creutzfeldt–Jakob disease with accumulations not only of abnormal prion protein (PrPTSE), but also three other proteins: hyperphosphorylated tau (p-tau), α-synuclein and ubiquitin; β-amyloid protein (Aβ) did not accumulate. Severity of brain lesions correlated with spongiform degeneration. No amyloid was detected. These results suggested that PrPTSE enhanced formation of p-tau and aggregation of α-synuclein and ubiquitin, but not Aβ, providing a new experimental model for neurodegenerative diseases associated with complex proteinopathies.

Most common neurodegenerative diseases feature deposition of protein amyloids and degeneration of brain networks. Amyloids are ordered protein assemblies that can act as templates for their own replication through monomer addition. Evidence suggests that this characteristic may underlie the progression of pathology in neurodegenerative diseases. Many different amyloid proteins, including Aβ, tau, and α-synuclein, exhibit properties similar to those of infectious prion protein in experimental systems: discrete and self-replicating amyloid structures, transcellular propagation of aggregation, and transmissible neuropathology. This review discusses the contribution of prion phenomena and transcellular propagation to the progression of pathology in common neurodegenerative diseases such as Alzheimer's and Parkinson's. It reviews fundamental events such as cell entry, amplification, and transcellular movement. It also discusses amyloid strains, which produce distinct patterns of neuropathology and spread through the nervous system. These concepts may impact the development of new diagnostic and therapeutic strategies.

The pathological aggregation of the presynaptic protein α-synuclein (α-syn) and propagation through synaptically coupled neuroanatomical tracts is increasingly thought to underlie the pathophysiological progression of Parkinson’s disease (PD) and related synucleinopathies. Although the precise molecular mechanisms responsible for the spreading of pathological α-syn accumulation in the CNS are not fully understood, growing evidence suggests that de novo α-syn misfolding and/or neuronal internalization of aggregated α-syn facilitates conformational templating of endogenous α-syn monomers in a mechanism reminiscent of prions. A refined understanding of the biochemical and cellular factors mediating the pathological neuron-to-neuron propagation of misfolded α-syn will potentially elucidate the etiology of PD and unravel novel targets for therapeutic intervention. Here, we discuss recent developments on the hypothesis regarding trans-synaptic propagation of α-syn pathology in the context of neuronal vulnerability and highlight the potential utility of novel experimental models of synucleinopathies.

Specific protein misfolding and aggregation are mechanisms underlying various neurodegenerative diseases such as prion disease and Alzheimer’s disease (AD). The misfolded proteins are involved in prions, amyloid-β (Aβ), tau, and α-synuclein disorders; they share common structural, biological, and biochemical characteristics, as well as similar mechanisms of aggregation and self-propagation. Pathological features of AD include the appearance of plaques consisting of deposition of protein Aβ and neurofibrillary tangles formed by the hyperphosphorylated tau protein. Although it is not clear how protein aggregation leads to AD, we are learning that the cellular prion protein (PrPC) plays an important role in the pathogenesis of AD. Herein, we first examined the pathogenesis of prion and AD with a focus on the contribution of PrPC to the development of AD. We analyzed the mechanisms that lead to the formation of a high affinity bond between Aβ oligomers (AβOs) and PrPC. Also, we studied the role of PrPC as an AβO receptor that initiates an AβO-induced signal cascade involving mGluR5, Fyn, Pyk2, and eEF2K linking Aβ and tau pathologies, resulting in the death of neurons in the central nervous system. Finally, we have described how the PrPC-AβOs interaction can be used as a new potential therapeutic target for the treatment of PrPC-dependent AD.

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.

Time-domain fluorescence lifetime imaging microscopy is presented for the detection of alpha-synuclein aggregation in neurons and for determining spread, thereby facilitating understanding of the development and progression of Parkinson’s disease.

Adrenaline (Adr) is generally considered as a full agonist able to induce in vitro the aggregation of human platelets and could play an important role in vivo in the appearance of thrombotic disorders when catecholamine levels are increased. Adr 2.5 M) induces the aggregation and secretion of 41 % of preloaded 3H-serotonin in human platelets in citrated plasma. This effect is not seen in plasma collected on 50 ATU/ml hirudin, and is due to the generation of traces of thrombin during blood collection and not to a direct effect of citrate itself, such asthe lowering of plasma free calcium. With washed human platelets suspended in Tyrode's buffer containing 2 mM Ca2+, 0.35 %albumin and apyrase, Adr (0.1 -100 M) doesnot cause shape change, aggregation or secretion of serotonin and does not modify platelet ultrastructure as judged by electron microscopy. Adr (1-100 M) does not change platelet membrane fluidity, as studied with the lipophilic fluorescent probe TMA-DPH. Adr has no direct effect on fibrinogen binding to intact platelets, intracellular Ca2+levels measured with quin2, or phosphorylation of 20 KDa or 47 KDapolypeptides, whereas all these parameters are modified after stimulation with ADP orthrombin. Adr potentiates the action of all types of aggregating agents on aggregation, secretion, intracellular Ca2+ levels,membrane fluidity, fibrinogen binding or protein phosphorylation. This effect is also seen with alpha2-adrenergic agonists (noradrenaline, alpha-methyl noradrenaline, clonidine) and is inhibited by alpha2-adrenergicantagonists such as yohimbine. The potentiation of platelet aggregation by Adr is not modified by prior incubation of the platelets with1mM aspirin for 15 min. This study shows that Adr alone does not induce modifications ofmorphology, metabolism or function of intactand functional washed human plateletsand that Adr cannot be considered per se as an aggregating agent. However, Adr interactswith alpha2-adrenergic receptors on human plateletsand potentiates biochemical and aggregatory responses induced by other platelet agonists.

The ability of cell surface receptor occupation to increase the activity of phospholipase A2 has been thought to be due to the prior activation of phospholipase C and an increase in the intracellular Ca2+ concentration. However, recent evidence from our and other laboratories has suggested that this may not be the case, but rather stimulation of phospholipase A2 may be under the control of separate receptor-activated events. We have investigated this further by comparing the ability of prostacyclin (PGI2) and epinephrine to alter platelet responses to thrombin and examining the resulting phospholipase A2 activities.Alpha-thrombin stimulated aggregation of human platelets, the formation of inositol phosphates and phosphatidic acid, mobilizaton of Ca2+ from internal stores and Ca2+ influx, protein phosporylation (47 kDa and 20 kDa) and arachidonic acid (AA) release. Each of these responses was partially inhibited by prostacyclin (PGI2) except that of AA release, which was abolished. In combination with epinephrine and PGI2, alpha-thrombin-induced aggregation, phosphatidic acid formation and protein phosphorylation were restored, but the release of AA only reached 50% of its control value. Epinephrine alone had no effect on any of these responses, either in the presence or absence of PGI2. Thus, alpha-thrombin-induced activation of phospholipase A2 is more sensitive to the effects of PGI2 than is phospholipase C, and supports the possibility that there are distinct control mechanisms for receptor activation of these enzymes. We are presently examining the role of Gs in the inhibition by PGI2 of platelet phospholipase A2 and of Gi in the thrombin stimulation of this enzyme