Dissertations / Theses on the topic 'Alpha synuclein'

Neurodegeneration, the progressive and irrevocable loss of neuronal structure, is quickly becoming an imposing health concern in a globally ageing society. While specific neurodegenerative conditions exhibit specific clinical symptoms and progressions, a common neuropathological feature is the misfolding, oligomerisation and fibrillation of certain proteins causing neuronal stress and death. Parkinson’s disease, PD, has long been characterised by the death of nerve cells focused in the substantia nigra pars compacta region of the midbrain and deposition of large protein aggregates, called Lewy Bodies, throughout the central nervous system. More recently, the protein which forms these inclusion bodies was identified as alpha synuclein, αSyn, a ubiquitous neuroprotein with no known function. Furthermore, persons with mutations in the SNCA gene, which codes for αSyn, exhibit PD progression at a far younger age with a more severe phenotype, positively linking αSyn with PD. αSyn is an intrinsically disordered protein, IDP, and generally persists as such in solution and inside bacterial and mammalian cells. However, when in contact with a lipid bilayer the protein will embed upon the surface in an amphipathic alpha helical conformation and can also aggregate, forming toxic oligomeric and fibrillar species containing significant β-sheet identity. Its function as a helical apolipoprotein and subcellular localisation to both the nucleus and synapse has led researchers to suggest that αSyn has a role synaptic transmission and release. However, knocking out the protein does not reduce viability or produce pathological abnormalities in neuronal structure. The helical form of the protein may also persist as transient, metastable helical bundles which are non-toxic and resist aggregation. While a number of studies and tools have been reported and developed to investigate the toxic oligomeric/fibrillar forms of αSyn, very little attention has been accorded to the helical conformation. This thesis will redress this balance by producing tools which will allow us to mimic the helical form of αSyn, promote the active refolding of the full-length protein using a stable, helical peptide template and produce antibodies which recognise helical αSyn specifically for use in discovery and chaperone-like refolding. In Chapter 2 a region of αSyn (14 amino acids) was identified with a unique primary sequence located within a mutation prone section of the protein. Peptide ‘stapling’ technologies were then employed using a panel of monosubstituted ‘staple’ diastereomers, to produce a highly helical portion of αSyn. Using several other protein targets particular diastereomeric ‘staple’ combinations were analysed for obvious trends in helical content. Using solution NMR, backbone refined three dimensional structures of these helical peptides were produced which showed that they were faithful structural homologues of their parent helical proteins. In Chapter 3 the drug-like properties and therapeutic potential of stable, helical αSyn peptides were investigated. Using fluorescently labelled peptide substrates, ‘stapled’ peptides were shown to be far more cell penetrant than their wild type equivalents and demonstrated that the mechanism for cellular uptake appears to be specific. Furthermore, under harsh proteolytic conditions the ‘stapled’, helical peptides were far more resistant to hydrolysis than wild type or ‘stapled’, poorly helical peptides. The ‘stapled’ peptides were also highly soluble and did not appear to aggregate in a time-dependent manner. Using ion mobility mass spectrometry, it was shown that incubation of full-length protein with the ‘stapled’, helical peptides caused a contraction in the hydrodynamic radius of the protein. However, using solution NMR no active refolding of αSyn was observed when under the same conditions. Rather small perturbations in chemical shift were apparent which did not suggest that the αSyn protein folded into a discrete structural conformation, such as an alpha helix. In Chapter 4 the stable, helical αSyn peptide was employed as a conformational model and unique antigen in antibody discovery. Immunisation with the ‘stapled’, helical αSyn peptide initially produced a pool of polyclonal antibodies with a half log specificity for the helical peptide. After bespoke affinity chromatography this was increased to three log orders of specificity. Initial immunocytochemistry did not detect any helical αSyn protein in SH-SY5Y cells. To validate the helical epitope on the full-length protein in vitro an assay based around flow cytometry of synthetic vesicle structures was developed, with their synthesis, characterisation and binding of the αSyn protein described.

Summary My PhD thesis is composed of two parts. A part deals with the characterization of alpha-synuclein (aS) dimers aggregation properties in respect to those of aS. The experimental work was conducted at CRIBI laboratory, at University of Padua, and constitutes the main project in which I was involved. During the third year of my PhD I spent six months at the Biopolymer Mass Spectrometry Laboratory of Imperial College in London. I conducted a glycomic analysis of mice tissues and a pilot study on expression and biosynthesis of mixed linked glucans emicellulose. Parkinson’s disease (PD) is a progressive, neurodegenerative disorder characterized by the loss of dopaminergic neurons in substantia nigra. The histological hallmarks of PD are intracellular inclusions, known as Lewy bodies (LBs), composed by filamentous and aggregated protein. The pathogenesis of the disease is still unclear, but a key step in the onset of PD is the aggregation of aS into amyloid fibrils, that deposit within LBs as the major component. Despite its importance in neurodegeneration, little is known about aS function, native physiological state and mechanism of aggregation. aS was recently described as a folded tetramer, but was generally considered a natively unfolded protein. aS is able to acquire alpha-helix conformation upon interaction with lipids and to convert to beta-structure in pathological processes. During the aggregation process, aS forms soluble oligomers, transient beta-structured intermediate between the physiological form of aS and amyloid fibrils. Dimerization of aS could represent a critical, rate-limiting step in the aggregation and amyloid formation of the protein. Therefore, we decided to study the aggregation of several different dimers of aS, produced through molecular biology techniques. A cysteine residue has been added at the N-terminal or at the C-terminal of aS, therefore producing a dimer N-N or C-C linked through a disulfide bond. A N-C dimer, formed two consecutive aS molecules, was obtained as a single polypeptide chain. During the project, another dimer, called DC dimer, was produced in order to further draw up the hydrophobic regions, and avoid the interferences of side chains within the molecule. DC is constituted by two consecutive central, highly amyloidogenic regions, containing aS residues from 1 to104 joined to residues from 29 to 140. The dimers represent a suitable tool for the study of intramolecular aS interaction pathway. Some remarkable differences define and limit the mobility freedom of the dimers respect to aS, hypothetically differentiating the fibrillation process of the four protein structures. The characterization of the dimers was performed using chemical and biophysical techniques in order to define their behaviour in solution as monomer. CD, IR and NMR spectroscopy studies show that all the dimers are unfolded. They undergo alpha-helical transition upon interaction with the detergent SDS. These results evidence that dimers strongly resemble aS conformational features. All the dimers were tested for the ability to form fibrils, by incubating the molecules under physiological buffer and at a protein concentration of 1 mg/ml. They show to be able to form fibrils, that are positive to Thioflavin T binding assay. Moreover, analysis of the structure of fibrils, conducted using circular dichroism (CD) and Fourier Transformed-IR (FTIR) spectroscopy, detects the structural transition from random to beta-sheet structure as attended for typical amyloid structure. Fibrils morphology was investigated by transmission electron microscopy (TEM) and atomic force microscopy (AFM) imaging. Fibrils derived from aS dimers are quite long, unbranched and formed by a single filament, a peculiar difference with aS fibril morphology. To identify which amino acids in the respective types of fibrils belong to the fibril core, proteolysis was performed. The rationale of this experiment reside in the fact that disordered regions of proteins are generally site of enzymatic attack and hydrolysis occurs at flexible chain region devoid of hydrogen-bonded secondary structure. Therefore, the prospects are to remove the flexible parts or tail from the amyloid core. Results showed that the core structures of the fibrils of the different molecules seems to be constituted by the same amino acidic region, which encompasses the segment 35-96, in analogy with previous studies. The kinetic of the process was analyzed by fluorescence techniques (ThT binding assay) and by evaluating the amount of protein present in fibrils on time. This calculation was indirectly performed measuring the absorbance of the supernatant obtained after centrifugation of each aliquot. NN and NC dimers show a slower kinetic of fibrillation than aS, while the rate of fibril formation of CC and DC dimers is faster than aS. Moreover, aggregation experiments on mixtures of aS in the presence of small amount of dimers were also conducted in order to check if the presence of dimer influence aS kinetic. Results evidenced the ability of CC dimer to affect the aggregation of aS. On the base of collected results, models of the dimer conformation within the fibrils are proposed. The research experience performed at Imperial College London gave me the possibility to learn and apply advanced techniques in mass spectrometry analysis of small organic compound, using GC-MS and MALDI-TOF spectrometers. N-acetylglucosaminyltransferase V (GlcNacT-V), encoded by the Mgat5 gene, is a medial Golgi enzyme which catalyzes the addiction of a beta-1,6-linked GlcNAc to the alpha-1,6 mannose of the trimannosyl N-glycan core. GlcNacT-V plays a pivotal role in the formation of tri- and tetra-antennary N-glycans on newly synthesized glycoprotein. This branch provides the preferred substrate for the enzymatic subsequent synthesis of polylactosamine chains and terminal modification including the Lewis antigens. In my study, glycomic analyses were performed to investigate possible changes in protein N-glycosylation in wild type conditions and in the absence of Mgat5 gene in C57B5 mice kidneys. In parallel, N-glycan profile of kidneys and spleens coming from mice treated with high fat diet GlcNAc supplementation were analyzed. Previous results demonstrate that the effects of GlcNAc salvage appear to increase flux to UDP-GlcNAc. Therefore we were interested to know whether this implementation affects N-glycan branching. Results show that Mgat5 deficient mouse kidney display less amount of tri-antennary and tetra-antennary structure compared to controls. However, GlcNAc dietary salvage has no apparent effect on N-linked glycosylation in the kidney and spleen, even if the experiments conducted on cell lines demonstrate that increased influx of UDP-GlcNAc resulted on increased N-glycan branching. Moreover, the performance of optimized glycome procedure allowed the identification of more tri-antennary glycan structures than the one reported on CFG (Consortium of Functional Glycomics) database.
Riassunto La mia tesi di dottorato è composta di due sezioni. Una sezione riguarda la caratterizzazione di dimeri di alpha-sinucleina (aS) in confronto con le proprietà di aS, sia in soluzione che in esperimenti di aggregazione. Il lavoro sperimentale è stato condotto nel laboratorio di Chimica delle Proteine (CRIBI Biotechnology Center), presso l’Università degli studi di Padova, e costituisce il progetto principale nel quale sono stata coinvolta. Durante il mio terzo anno di dottorato ho trascorso 6 mesi al laboratorio Biopolymer Mass Spectrometry Laboratory presso l’Imperial College a Londra. In questo laboratorio sono stata coinvolta in due progetti: uno studio di analisi glicomica di tessuti murini e un progetto pilota sulla biosintesi di emicellulosa mixed linked glucans (MLG). Il morbo di Parkinson è una malattia neurodegenerativa progressiva caratterizzata dalla perdita di neuroni dopaminergici nella substantia nigra. La principale caratteristica istologica della malattia è la presenza di inclusioni intracellulari, conosciute come corpi di Lewy, composti da aggregati proteici filamentosi. La patogenesi della malattia è ancora poco chiara, ma un passaggio chiave nello sviluppo della malattia è l’aggregazione di alpha-synuclein (aS) in fibrille amiloidi, che si accumulano dei corpi di Lewy e ne costituiscono il componente principale. Nonostante la sua importanza nella neurodegenerazione, si conoscono poco la funzione di aS, il suo stato nativo fisiologico e il meccanismo di aggregazione. aS è stata di recente descritta come un tetramero di proteine in alpha-elica, ma aS è stata generalmente descritta come una proteina natively unfolded. aS assume conformazione ad alpha-elica a seguito di interazione con lipidi e converte a struttura beta durante i processi patologici. Durante il processo di aggregazione, aS forma oligomeri solubili di struttura beta, transienti intermedi tra la forma fisiologica di aS e le fibrille amiloidi. La dimerizzazione di aS può rappresentare un fattore limitante nell’aggregazione e nella formazione di struttura amiloide. Pertanto, abbiamo deciso di studiare l’aggregazione di diversi dimeri di aS, prodotti mediante biologia molecolare. E’ stato aggiunto un residuo di cisteina all’ N- o al C- terminale di aS, producendo quindi dimeri NN o CC, legati attraverso un legame disolfuro. Un dimero NC, formato da due molecole consecutive di aS, è stato ottenuto come singola catena polipeptidica. Durante il progetto è stato prodotto un altro dimero, chiamato DC, disegnato in modo da avvicinare ulteriormente le regioni idrofobiche di aS, ed evitare le interferenze provocate dalle catene laterali, che vengono a trovarsi all’interno della molecola nei dimeri NN, CC ed NC. Il dimero DC contiene i residui 1-104 uniti al segmento 29-140 di aS, ed è quindi costituito da due regioni centrali di aS, altamente amilodoigeniche, disposte in modo consecutivo. I dimeri rappresentano uno strumento adatto per lo studio delle interazioni intramolecolari di aS. Alcune differenze sostanziali definiscono e limitano la libertà di movimento dei dimeri rispetto ad aS, ipoteticamente differenziando il processo di fibrillazione delle cinque strutture proteiche. La caratterizzazione dei dimeri è stata effettuata utilizzando tecniche biofisiche e chimiche al fine di definire il loro comportamento in soluzione come monomero. Studi di dicroismo circolare (CD), spettroscopia IR ed NMR hanno dimostrato che tutti i dimeri sono unfolded. Tutti effettuano transizione ad alpha-elica a seguito dell’interazione con il detergente SDS. Questi risultati provano che i dimeri hanno caratteristiche conformazionali simili ad aS. Successivamente, è stata esaminata la capacità dei dimeri di formare fibrille, incubando le molecole in tampone fisiologico alla concentrazione di 1 mg/ml. Tiutti sono in grado di formare fibrille, che sono positive al saggio di legame alla Tioflavina T (ThT), generalmente utilizzato per determinare la presenza di struttura amiloide. Inoltre, le analisi della struttura delle fibrille, condotte usando CD e spettroscopia IR in trasformata di Fourier (FT-IR), rilevano la presenza di transizione strutturale da random a struttura beta come ci si aspetta per fibrille amiloidi. La morfologia delle fibrille è stata studiata mediante microscopia elettronica a trasmissione (TEM) e microscopia di forza atomica (AFM). Le fibrille derivate dai dimeri di aS sono abbastanza lunghe, non ramificate e a singolo filamento, una differenza peculiare rispetto alle fibrille di aS, che si presentano twisted e formate da più filamenti. Per identificare quali amminoacidi di ciascun dimero fosse coinvolto nel core fibrillare sono sati eseguiti esperimenti di proteolisi. Il razionale di questo esperimento risiede nel fatto che le regioni non strutturate delle proteine sono in genere sito di attacco enzimatico, e l’idrolisi si verifica quindi in regioni flessibili, sprovviste di legani idrogeno intermolecolari che stabilizzano una struttura secondaria. Quindi lo scopo dell’esperimento è di rimuovere le parti flessibili dal core amyloide. I risultati hanno mostrato come le strutture core delle fibrille dei diversi dimeri sembrino essere costituite dalla stessa regione amminoacidica, che comprende il segmento 35-96, in analogia con studi precedenti su aS. La cinetica del processo è stata analizzata con tecniche di fluorescenza (saggio ThT) e valutando la quantità di proteine presenti nel tempo. Questo calcolo è stato effettuato indirettamente misurando l’assorbanza del surnatante ottenuto dopo ultracentrifugazione delle aliquote prelevate da miscele di aggregazione a diversi tempi. I dimeri NN ed NC hanno mostrato una cinetica di aggregazione più lenta rispetto ad aS, mentre il tasso di formazione delle fibrille di CC e DC è più veloce. Inoltre, esperimenti di aggregazione su miscele di aS in presenza di piccole quantità di dimeri sono stati condotti al fine di verificare se la presenza del dimero influenzasse la cinetica di aS. I risultati hanno evidenziato la capacità del dimero CC di influenzare l’aggregazione di aS. Sulla base dei risultati ottenuti, sono stati proposti dei modelli sulla conformazione dei dimeri all’interno delle fibrille. L’esperienza di ricerca svolta all’Imperial College London mi ha dato la possibilità di imparare e applicare tecniche avanzate di spettrometria di massa (MS) sull’analisi di composti organici, utilizzando gas cromatografia accoppiata ad MS (GC-MS) e spettrometri MALDI-TOF. L’enzima N-acetylglucosaminyltransferase V (GlcNAc-V), codificato dal gene Mgat 5, è un enzima del Golgi che catalizza l’addizione di un GlcNAc in posizione beta-1,6 a un mannosio alpha-1,6 della struttura di base degli zuccheri legati a residui amminici (N-glicani). GlcNAc-V svolge un ruolo fondamentale nella formazione di N-glicani a tre- e quattro-antenne su una proteina appena glicosilata. Queste ramificazioni forniscono il substrato favorito per la successiva sintesi enzimatica di catene poli-lactosamminiche e per le modificazioni terminali, compresi gli antigeni di Lewis. Ho svolto analisi glicomiche su tessuti renali murini per studiare possibili cambiamenti nella N-glicosilazione in topi wild type e knock out per il gene Mgat 5. In parallelo, è stato analizzato il profilo glicomico di tessuti renali e di milza di topi alimentati con una dieta ricca di GlcNAc. Risultati precedenti avevano dimostrato un aumento nel flussio di UDP-GlcNAc (substrato di GlcNAc-V), perciò eravamo interessati a determinare se il maggione apporto di zucchero influenzasse le glicosilazioni proteiche. I risultati hanno evidenziato come le glicoproteine dei topi ko per Mgat 5 hanno meno strutture a tre- e quattro-antenne nelle glicosilazioni rispetto ai controlli. L’apporto di GlcNAc nella dieta non ha alcun affetto apparente sulla struttura e composizione delle glicosilazioni dei tessuti analizzati, nonostante precedenti esperimenti condotti su linee cellulari abbiano avuto un diverso esito. Inoltre, le analisi che ho condotto hanno permesso di identificare glicosilazioni non ancora registrate nel database CFG (Consortium of Functional Glycomics) per i tessuti analizzati.

To date there is no accepted clinical diagnostic test for Parkinson's disease (PD) based on biochemical analyses of blood or cerebrospinal uid. Currently, diagnosis, measurement of disease progression and response to therapeutic intervention are based on clinical observation, but the rst neuronal dysfunction precede the earliest recognition of symptom by at least 5 - 10 years. A potential diagnostic biomarker is oligomeric alpha-synuclein which in recent papers have reported a signicant quantitative dierence between PD and controls. In this master thesis, a method for measuring oligomeric levels of alpha-synuclein is presented together with a monomeric measuring commercial kit used to measure alpha-synuclein in a preclinical model of PD. A signicant dierence of monomeric levels could be detected between two weeks and four weeks post injection of a vector containing the gene for human alpha-synuclein, no signicant dierence between four and eight weeks was found.

Parkinson’s disease is a common neurological disorder with a complex etiology. The disease is characterized by a progressive loss of dopaminergic cells in the substantia nigra, which leads to motor function and sometimes cognitive function disabilities. One of the pathological hallmarks in Parkinson’s disease is the cytoplasmic inclusions called Lewy bodies found in the dopamine neurons. The aggregated protein α-synuclein is a main component of Lewy bodies. In view of severe symptoms and the upcoming of problematic side effects that are developed by the current most commonly used treatment in Parkinson’s disease, new treatment strategies need to be elucidated. One such strategy is replacing the lost dopamine neurons with new dopamine-rich tissue. To improve survival of the implanted neurons, neurotrophic factors have been used. Glial cell line-derived neurotrophic factor (GDNF), which was discovered in 1993, improves survival of ventral mesencephalic dopamine neurons and enhances dopamine nerve fiber formation according to several studies. Thus, GDNF can be used to improve dopamine-rich graft outgrowth into the host brain as well as inducing sprouting from endogenous remaining nerve fibers. This study was performed on Gdnf gene-deleted mice to investigate the role of GDNF on the nigrostriatal dopamine system. The transplantation technique was used to create a nigrostriatal microcircuit from ventral mesencephalon (VM) and the lateral ganglionic eminence (LGE) from different Gdnf gene-deleted mice. The tissue was grafted into the lateral ventricle of wildtype mice. The results revealed that reduced concentrations of GDNF, as a consequence from the Gdnf gene deletion, had effects on survival of dopamine neurons and the dopamine innervation of the nigrostriatal microcircuit. All transplants had survived at 3 months independently of Gdnf genotype, however, the grafts derived from Gdnf gene-deleted tissue had died at 6 months. Transplants with partial Gdnf gene deletion survived up to 12 months after transplantation. Moreover, the dopaminergic innervation of striatal co-grafts was impaired in Gdnf gene-deleted tissue. These results highlight the role of GDNF for long-term maintenance of the nigrostriatal dopamine system. To further investigate the role of GDNF expression on survival and organization of the nigrostriatal dopamine system, VM and LGE as single or combined to double co-grafts created from mismatches in Gdnf genotypes were transplanted into the lateral ventricle of wildtype mice. Survival of the single grafts was monitored over one year using a 9.4T MR scanner. The size of single LGE transplants was significantly reduced by the lack of GDNF already at 2 weeks postgrafting while the size of single VM was maintained over time, independently of GDNF expression. The double grafts were evaluated at 2 months, and the results revealed that lack of GDNF in LGE reduced the dopamine cell survival, while no loss of dopamine neurons was found in VM single grafts. The dopaminergic innervation of LGE was affected by absence of GDNF, which also caused a disorganization of the striatal portion of the co-grafts. Small, cytoplasmic inclusions were frequently found in the dopamine neurons in grafts lacking GDNF expression. These inclusions were not possible to classify as Lewy bodies by immunohistochemistry and the presence of phospho-α-synuclein and ubiquitin; however, mitochondrial dysfunction could not be excluded. To further study the death of the dopamine neurons by the deprivation of GDNF, the attention was turned to how Lewy bodies are developed. With respect to the high levels of α-synuclein that was found in the striatum, this area was selected as a target to inject the small molecule – FN075, which stimulates α-synuclein aggregation, to further investigate the role of α-synuclein in the formation of cytoplasmic inclusions. The results revealed that cytoplasmic inclusions, similar to those found in the grafts, was present at 1 month after the injection, while impairment in sensorimotor function was exhibited, the number of dopamine neurons was not changed at 6 months after the injection. Injecting the templator to the substantia nigra, however, significantly reduced the number of TH-positive neurons at 3 months after injection. In conclusion, these studies elucidate the role of GDNF for maintenance and survival of the nigrostriatal dopamine system and mechanisms of dopamine cell death using small molecules that template the α-synuclein aggregation.

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. It is characterized by the presence of intracellular inclusions termed Lewy bodies (LBs) and Lewy neuritis (LNs) in the brain, in which α-Syn aggregates constitute the main component. Therefore, α-Syn aggregation was implicated in the pathogenesis of PD. Structurally α-Syn is a disordered protein with little ordered structure under physiological conditions. However, research of α-Syn has provided substantial information about its structural properties. The precise function of α-Syn is still under investigation. Research has also shown that metals, such as copper and iron, accelerate α-Syn aggregation and fibrillation in vitro and are proposed to play an important role in vitro. In this study, isothermal titration calorimetry was used to determine iron binding properties to α-Syn revealing the presence of two binding sites for iron with an affinity of 1.06 x 105 M-1 and a dissociation constant of ~ 10μM which is physiologically relevant to iron content in the brain. In addition, α-Syn was found to reduce iron in the presence of copper. This property was demonstrated via ferrozine based assay. In vitro, thoflavin-T fluorescence assay was used to investigate the mechanism by which metals induce α-Syn aggregation and whether it is related to metal binding. Metals, mainly copper and iron, caused 2-fold increase in the aggregation rate of WT α-Syn and its metal binding mutants. Linking that to the increased metal content in the brain, α-Syn aggregation can cause changes in tissue composition, thus altering the normal functional environment in the brain. Moreover, western blotting analysis showed that copper increases the aggregate formation in mammalian dopaminergic cells over-expressing α-Syn.

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.

α-Synuclein (αSyn) is highly abundant cytosolic protein whose conversion into insoluble fibrils is a pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Despite decades of research, fundamental questions regarding αSyn biology are unresolved. Soluble, prefibrillar oligomers, not their fibrillar end products, are believed to be neurotoxic in humans and in disease models, but their mechanism of action remains unknown. Evidence from our lab and others increasingly suggests that, in healthy cells, αSyn does not exist purely as an unfolded monomer, as the field has long believed, but also as aggregation-resistant, α-helical oligomers; however, their physiological role remains controversial. Thus, my aim was twofold: to characterize toxic αSyn species in the context of mitochondrial dysfunction, a central phenotypic feature of PD; and to purify helical αSyn oligomers from human brain to enable further characterization of physiological αSyn.

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2009.
Title from electronic title page (viewed Oct. 5, 2009). "... in partial fulfillment of the requirements for the degree of Doctorate of Philosophy in the Department of Chemistry." Discipline: Chemistry; Department/School: Chemistry.

L'agrégation d'alpha-synucléine et la propagation de ces agrégats de neurone à neurone ont, de manière récurrente, été montrées comme étant au cœur du processus physiopathologique de différentes maladies neurodégénératives telles que la maladie de Parkinson, les démences à corps de Lewy ou les atrophies multisystématisées. Bien que ces synucléinopathies aient en commun la présence de dépôts fibrillaires riches en alpha-synucléine, les phénotypes pathologiques sont différents. Notre hypothèse est que la présence de différentes "souches" d'alpha-synucléines fibrillaires présentant différentes affinités, tropismes, propriétés d'internalisation et de propagation pourrait expliquer l'hétérogénéité physiopathologique et clinique retrouvée dans la maladie de Parkinson et les autres synucléinopathies. Le but de ce projet était de déterminer comment interférer avec la liaison et la propagation "prion-like" de ces assemblages d'alpha-synucléines dans les neurones. Dans cet objectif, nous avons caractérisé les surfaces de trois souches d’alpha-synucléine. Dans un premier temps, nous avons mis en place des approches in vitro de protéolyses ménagées et de marquages hydrogène-deutérium combinées à la spectrométrie de masse qui nous ont permis de cartographier la surface d'agrégats fibrillaires d'alpha-synucléines générés in vitro. Dans un second temps, nous avons étudié la prise charge de différentes souches d’alpha-synucléine par des cultures primaires de neurones ou d’astrocytes afin d’analyser les caractéristiques de dégradation des souches in cellulo, liées à leurs spécificités de surface déterminées préalablement. La cartographie réalisée ouvre la voie, à long terme, au développement d’outils diagnostics ou thérapeutiques hautement spécifiques capables de reconnaitre spécifiquement différentes souches d’alpha-synucléine ou d’inhiber la propagation de cellule à cellule de ces agrégats afin de ralentir ou d'arrêter la progression de la maladie
The aggregation of alpha-synuclein and the spread of aggregates from neuron to neuron have been consistently shown to be at the heart of the pathophysiological process of devasting neurodegenerative diseases like Parkinson's disease, dementia with Lewy bodies or multiple system atrophy. While fibrillar alpha-synuclein rich deposits are a common hallmark of synucleinopathies, distinct pathological phenotypes are observed. We hypothesize that different "strains" of fibrillar alpha-synuclein with different affinity/tropism, internalization and seeding properties, may account for the patho-physiological and clinical heterogeneity in Parkinson's disease and other synucleinopathies. We aim to determine a way to interfere with the binding to neurons of distinct alpha-synuclein assemblies and their prion-like propagation. To this end, we mapped the surface of three distinct alpha-synuclein strains. First, we implemented limited proteolysis and hydrogene-deuterium approaches combined to mass spectrometry in order to map, in vitro, the solvent exposed-surfaces of fibrillar alpha-synuclein assemblies generated in vitro. Second, we studied the processing of different alpha-synuclein strains using primary cultures of neurons and astrocytes in order to analyze the strain degradation characteristics in cellulo, related to the surface specificities determined in vitro. Mapping the surfaces of those assemblies and identification of exposed and protected strain-specific sequences open the way, in the long term, for developing highly specific binders that might either detect specific alpha-synuclein strains or inhibit cell-to-cell propagation disease progression

alpha-synucleinopathies are a group of neurodegenerative disorders characterized by the presence of abnormally aggregated alpha-synuclein (aS). Recent evidence suggests that the early site of aS aggregation is synapses, where aS seems to play its physiological role. Moreover, aggregated aS is reported to be secreted by cells, suggesting its potential involvement in disease initiation and progression. Considering the nature of neurodegenerative disorders as well as the defined, step-wise spreading of Lewy body pathology in alpha-synucleinopathies, the idea of extracellular aS as a pathogenic prion-like agent is extremely appealing. This research project developed in this frame and it is focused on the propagation of aS toxic species mediated by a particular type of extracellular vesicles, exosomes. To this aim, exosomes containing aS and DOPAL modified aS oligomers were purified by HEK293T cells and we focused on the effect of these vesicles on different cell types. Since, in neurons, exosomes appear to be secreted in a spatially and regulated manner through synapses, we first investigate their effect on primary neuronal culture synapses . Upon incubation, aS containing exosomes and, more significantly, DOPAL-modified aS containing exosomes are delivered to synapses, where they alter proteins amounts, function and neuronal morphology. aS containing exosomes contribute also to neuroinflammation: they mediate increment of IL-1beta cytokine production in mouse immortalized microglia cells. Our results highlight an exosomes-driven toxicity of aS not only to neuronal synapses, but also to microglia, inducing the secretion of IL-1beta. Therefore, aS containing exosomes appear as a vehicle of aS toxicity, which might be interesting not only as a future therapeutic target, but also as a potential biomarker for alpha-synucleinopathies.
Le alpha-sinucleinopatie sono un gruppo di malattie neurodegenerative caratterizzate dall'anormale aggregazione della proteina alpha-Sinucleina (aS). La localizzazione dell'aS è prevalentemente pre-sinaptica, ove sembra non solo svolgere la propria funzione fisiologica, ma anche iniziare l'alterazione patologica della sua struttura. Nonostante i meccanismi alla base di questo evento non siano noti, una delle ipotesi proposte è l'internalizzazione di specie tossiche di aS rilasciate da altre cellule. Queste forme di aS, infatti, al pari di quello che avviene nelle malattie prioniche, indurrebbero l'aggregazione dell'aS endogena. Tali premesse hanno indotto uno studio principalmente focalizzato sull'impatto a livello sinaptico di specie tossiche di aS veicolate dagli esosomi. Gli esosomi sono stati purificati da cellule HEK293T trasfettate con aS-EGFP e trattate o meno con il DOPAL, un metabolita tossico della dopamina che è in grado di indurre l'aggregazione dell'aS. Una volta verificato che le vescicole contenessero specie aggregate di aS, queste sono state poi incubate con culture neuronali primarie. Per valutare il loro effetto a livello sinaptico sono stati presi in considerazione vari fattori. Per primo è stata dimostrata una riduzione dei livelli di sinaptofisina e PSD-95, due proteine marker rispettivamente della pre- e della post-sinapsi. Queste alterazioni sono anche accompagnate da una disfunzione a livello sinaptico, caratterizzata non solo da una diminuzione del numero di vescicole per sinapsi, ma anche da una loro maggiore distanza dalla zona attiva. Anche la morfologia neuronale è stata alterata mentre non si è registrato alcun aumento di marker necrotici o apoptotici. Gli esosomi contenenti aS e aS modificata da DOPAL sono stati poi incubati con cellule di microglia primaria al fine di valutare se erano in grado di indurre una risposta infiammatoria. Il trattamento con gli esosomi ha aumentato la concentrazione della citochina pro-infiammatoria IL-1beta nel medium, facendo ipotizzare un loro coinvolgimento anche nella neuro-infiammazione. In conclusione questi dati suggeriscono che gli esosomi rilasciati dalle cellule e contenenti specie aggregate di aS propaghino la tossicità  a livello neuronale e stimolino nella microglia la produzione di fattori pro-infiammatori, creando una sorta di circolo vizioso che ne aumenta l'effetto patologico. Gli esosomi contenenti specie aggregate di aS potrebbero quindi non solo diventare nuovi target terapeutici, ma anche potenziali biomarker per la diagnosi delle alfa-sinucleinopatie.

Genetic studies have revealed three mutations (A30P, A53T and E46K) in alpha-synuclein (alpha-Syn) that cause Parkinson's disease (PD) in a small number of pedigrees with autosomal dominant inheritance. For the purpose of this thesis an in vitro model has been developed by stably over-expressing wild type (wt), A30P or A53T mutant alpha-Syn in ND7 neuronal cells. Wt alpha-Syn can enhance cell death in response to ischaemia/reoxygenation or staurosporine treatment whilst protecting against serum removal and dopamine-induced cell death in this system. In contrast, both mutant forms of alpha-Syn enhance cell death. The above stresses were used to induce primarily apoptotic cell death, implicated in PD pathology. Hence, the PD-associated mutations convert alpha-Syn from a protein which could modulate cell death differently in different circumstances to forms which are deleterious in response to various stresses. Subsequently, the neuroprotective effect of various heat shock proteins (hsps) in the above system was studied, utilising a Herpes Simplex Virus-based gene delivery system. For the first time, it was demonstrated that in an in vitro mammalian model of alpha-Syn-induced toxicity over-expression of hsp27 protects, under all the stresses tested, both wt and mutant alpha-Syn expressing cells, as assessed by multiple apoptotic/necrotic death assays. Interestingly, A30P alpha-Syn expressing cells were markedly protected by caspase-8 and caspase-9 inhibition as well as by hsp27 over-expression. No synergy between hsp27 and the caspase inhibitors was observed. In addition, hsp70 conferred protection only to wt alpha-Syn expressing cells exposed to ischaemia whereas hsp56 had no protective role in this system. Hence, hsp27 was neuroprotective by interfering with the enhanced caspase-dependent cell death resulting from mutant A30P alpha-Syn over-expression. Finally, studies of the mitochondrial status in this system were performed to further explore the site of action of hsp27. Hsp27 reduced significantly the mitochondrial membrane potential loss in stressed A30P mutant alpha-Syn cells and this correlates well with their enhanced cell survival. These findings suggest that hsp27 has a novel neuroprotective role against mutant alpha-Syn toxicity and this is achieved by interfering with the caspase cascade and mechanisms modulating the mitochondrial membrane potential.

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.

In this observational cross-sectional study, using an immunoenzymatic technique we assayed and compared total plasma alpha-synuclein concentrations in 69 patients with Parkinson's disease and 110 age-matched healthy control subjects. Two previously unreported findings concerned gender. First, plasma alpha-synuclein concentrations measured in the more advanced parkinsonian disease stages decreased in men but not in women. Second, again only in men, plasma alphasynuclein concentration was associated with cognitive impairments, hallucinations, depressed mood and sleep disorders. These findings underline the gender-related differences in parkinsonian patients and indicate plasma alpha-synuclein expression as a potential biological marker for Parkinson's disease progression in men.

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.

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.

Misfolding and aggregation of the synaptic protein alpha-synuclein (αsyn) is, at present, considered one of the main drivers of the pathogenesis of Parkinson’s disease (PD). PD is a complex neurodegenerative disorder characterized by progressive loss of DAergic neurons in the substantia nigra pars compacta (SNpc) and deposition of insoluble proteinaceous inclusions containing αsyn, called Lewy Bodies (LB), in different regions of the nervous system. Toxic species of αsyn were demonstrated to affect multiple cellular pathways, mediating synaptic dysfunction even before a dramatic loss of SNpc neurons has occurred. Besides its neurotoxicity towards the DAergic system, αsyn has been recently reported to affect also the corticostriatal glutamatergic signalling, modulating the synaptic levels and activity of NMDA-type glutamate receptors. However, the precise molecular events underlying αsyn synaptic toxicity are still elusive. The synaptic retention of NMDARs was recently demonstrated to be strictly correlated to the interaction with the protein Rabphilin-3A (Rph3A). Interestingly, alterations of Rph3A expression and its interaction with NMDARs at the corticostriatal synapse have been described in advanced PD stages. Besides, a direct αsyn/Rph3A interaction, modulated in presence of LB, has been put forward. Starting from these previous findings, this PhD project is aimed at characterizing Rph3A role in the early stages of PD to identify pharmacological approaches able to slow down αsyn-induced synaptic toxicity. Exploiting different imaging and biochemical approaches, Rph3A was confirmed as a novel αsyn interactor at synaptic sites. In addition, using an in vivo mouse model of αsyn-induced PD, we found that oligomers and fibrils (PFF) of αsyn reduced AMPAR and NMDAR subunits at the striatal excitatory synapse, prior to cause significant DAergic neurodegeneration. At the same time point, αsyn-mice showed decreased striatal dendritic spine density and early motor impairments. Interestingly, I also found that αsyn oligomers and PFF selectively reduced striatal Rph3A postsynaptic levels and Rph3A binding to GluN2A subunit of NMDAR, suggesting Rph3A and Rph3A/αsyn complex as possible mediators of the synaptic dysfunction. Based on these hypothesis, modulatory strategies aimed at restoring Rph3A synaptic levels were firstly tested in an in vitro model of PFF-induced synaptopathy. In particular, a Rph3A/αsyn uncoupling compound (Compound B), identified through a bioinformatic screening, revealed efficacious in blocking spine loss caused by PFF exposure. In the same experimental model, synaptic defects were also prevented by Rph3A overexpression. Based on these in vitro results, these Rph3A-modulatory approaches were then investigated using the αsyn-induced PD mouse model. Notably, chronic intracerebroventricular treatment with Compound B confirmed its efficacy in blocking αsyn-dependent decrease in striatal spine density. Furthermore, striatal delivery of an AAV overexpressing Rph3A resulted sufficient in preventing the early motor impairments found in αsyn-lesioned mice. In conclusion, the results of this PhD thesis demonstrate that Rph3A and Rph3A/αsyn complex significantly contribute to the molecular events underlying early dysfunction of the striatal glutamatergic synapse in PD, therefore representing novel and promising pharmacological targets.

Positron emission tomography (PET) is a non-invasive medical imaging technique that allows visualisation and quantification of biochemical, physiological and pharmacological processes in living subjects. This is achieved through application of radiotracers – compounds labelled with positron emitting radionuclides. Neurodegeneration is the progressive loss of neurons resulting in impairment of brain function leading to cognitive decline and can affect movement. The underlying pathology of many neurodegenerative diseases is misfolding of proteins such as α-synuclein, the key pathological hallmark of Parkinson’s disease. Also implicated in the processes of neurodegeneration is neuroinflammation, which is observed by the activation of microglia – the immune cells of the brain. Activation of microglia is associated with the upregulation of the 18 kDa mitochondrial translocator protein (TSPO). This work has involved the synthesis and characterisation of novel compounds that have the potential for being applied as radiotracers for imaging α-synuclein fibrils (project 1), or TSPO (project 2) via PET. Over the course of project 1 a library of compounds was synthesised based upon structural modifications of a lead structure identified from the literature. These compounds then underwent screening via biophysical methodologies in order to determine their affinity to α-synuclein fibrils. This stage of the work involved the development of a novel biophysical technique – microscale thermophoresis (MST). A general automated radiosynthetic method to afford the [18F]fluoro-derivatives of these compounds has also been developed, and preliminary in vitro autoradiography studies and an in vivo microPET scan has been performed. For project 2, an automated radiosynthetic method was developed to produce [18F]GE387, a lead compound identified through collaboration with GE Healthcare. This radiotracer has then been applied to preliminary in vitro autoradiography and an in vivo microPET study using rats with induced neuroinflammation alongside control rats.

α-Synuclein (α-syn) plays a central role in the pathogenesis of neurodegenerative disorders collectively known as “synucleinopathies” that include Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Understanding the underlying molecular mechanisms of neurodegenerative diseases is indispensably important because of the prevalence of these devastating conditions in the elderly population. Several findings from cell culture and in vivo experiments suggest intercellular transfer of α-syn aggregates. The concept of intracellular α-syn pathology spread was recently extended by the discovery of propagation of α-syn aggregates throughout PD brains. The resulting concept of cell-to-cell propagation of α-syn pathology comprises of its release, uptake, and subsequently seeding of intracellular α-syn aggregation in recipient cells. α-Synuclein (α-syn) plays a central role in the pathogenesis of neurodegenerative disorders collectively known as “synucleinopathies” that include Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Understanding the underlying molecular mechanisms of neurodegenerative diseases is indispensably important because of the prevalence of these devastating conditions in the elderly population. Several findings from cell culture and in vivo experiments suggest intercellular transfer of α-syn aggregates. The concept of intracellular α-syn pathology spread was recently extended by the discovery of propagation of α-syn aggregates throughout PD brains. The resulting concept of cell-to-cell propagation of α-syn pathology comprises of its release, uptake, and subsequently seeding of intracellular α-syn aggregation in recipient cells. In this PhD thesis the methodology used to obtain synthetic mammalian prions has been used to obtain recombinant human and mouse α-syn amyloids in order to characterize whether synthetic material can promote prion-like accumulation of its soluble counterpart in neuronal cell lines in vitro and in wild type (WT) mice in vivo. To address these hypotheses, in the first part of the work the human neuroblastoma SH-SY5Y cell line and WT CD-1 mice were used. A single exposure to human α-syn amyloid fibrils was sufficient to induce aggregation of endogenous α-syn in SH-SY5Y cells. Remarkably, endogenous WT α-syn was sufficient for aggregate formation and overexpression of the protein was not required. Our results provide compelling evidence that endogenous α-syn can accumulate in cell culture after a single exposure to exogenous α-syn short amyloid fibrils. Importantly, using α-syn short amyloid fibrils as seed, endogenous α-syn aggregates and accumulates over several passages in cell culture. We further analyzed this phenomenon in vivo in WT CD-1 mice, where intracerebral inoculation of synthetic α-syn short amyloid fibrils induced the formation of phosphorylated α-syn aggregates in CNS regions distinct from the injection site. After this observation, further studies were performed in order to understand the mechanism of internalization of α-syn amyloids. Indeed, while the function of the cellular prion protein (PrP C) is still under debate, several reports indicate that PrPC interacts with amyloid form proteins, Aβ oligomers and PrP scrapie (PrPSc) fibrils. In order to investigate α-Syn amyloid accumulation and its interaction with prion protein this, we used the same technique as in the first part of our study, working on mouse α-syn protein. Here, we explored the uptake of α-syn amyloids in a neuroblastoma cell line: N2a cells which endogenously express PrPC (N2a), cells knocked out for PrPC (N2a KO), N2a KO where the PrP was re-introduced using transfection protocol (N2a PrPFL), and scrapie infected N2a (ScN2a) cells. Our results show that the uptake of α-syn amyloids is lower in N2a KO compared to control cells (N2a), raising the possibility that PrPmediates the uptake of α-syn amyloids within cytoplasm of N2a cells. Subsequently, biochemical characterization of this putative interaction through the use of two distinct cross-link molecules has been investigated. In the prion replication model, direct interaction between PrPSc and endogenous PrPC is required for the formation of further infectious prions. In fact, molecules that specifically bind cellular PrPC may interrupt the production of prions. To further analyze the effect of the addition of recombinant α-syn amyloids to scrapie infected cells, residual PK-resistant levels of PrP were investigated. ScN2a cells exposed to recombinant α-syn amyloids displayed drastically reduced levels of PK-resistant PrPSc. This observation supports the hypothesis that cell surface PrPC might directly bind α-syn amyloids as previously suggested. Finally, further work was required to validate the importance of this interaction in disease progression in vivo. Thus, stereotaxic injections of α-syn amyloids in substantia nigra pars compacta and striatum in FVB PrPWT and FVB PrPKO mice were performed. In conclusion, our findings suggest a role for PrP C in the regulation of α-syn uptake, thus evidencing a link between the two neurodegeneration associated proteins. Moreover, this study presents new insight on the possible implication of the prion protein in Parkinson’s disease. phenomenon in vivo in WT CD-1 mice, where intracerebral inoculation of synthetic α-syn short amyloid fibrils induced the formation of phosphorylated α-syn aggregates in CNS regions distinct from the injection site.

Thesis: Ph. D. in Physical Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2015.
Cataloged from PDF version of thesis. In title on title page, "[alpha]" appears as lower case Greek letters. Vita.
Includes bibliographical references (pages 91-104).
Parkinson's disease (PD) affects over 10 million people worldwide and has no cure. Moreover, current treatments for PD have limited efficacy. Studies that advance our understanding of the mechanism of neurodegeneration in PD will provide guidance in our search for effective therapies for this neurodegenerative disorder. PD is characterized clinically by motor deficits - namely resting tremors, rigidity, bradykinesia and postural instability - and pathologically by intraneuronal inclusions in the substantia nigra. Several studies suggest that a-synuclein, the major component of these intracellular inclusions, plays a major role in the neurodegenerative process. Therefore understanding the structural properties of [alpha]-synuclein and its aggregation mechanism is of particular interest. [alpha]-synuclein is particularly challenging to study because it is an Intrinsically Disordered Protein (IDP); i.e., it lacks a well-defined structure in aqueous solution. Unlike folded proteins, IDPs typically interconvert between many different conformations during their biological lifetime. In this thesis we apply novel methods to develop models for IDPs and apply them to asynuclein. The overriding hypothesis that forms the basis of this work is that IDPs in solution can be modeled as a finite set of energetically favorable structures, where each structure corresponds to an energy minimum on a complex energy landscape. The number of structures in the resulting ensemble is related to the resolution in which one wishes to view the energy landscape of the protein. We demonstrate that this approach leads to new insights into the aggregation mechanism of [alpha]-synuclein.
by Orly Ullman.
Ph. D. in Physical Chemistry

Parkinson’s disease (PD) is the second most common neurodegenerative condition to affect humans, and is characterised by the loss of dopaminergic neurons from the substantia nigra pars compacta (SNpc) in the midbrain along with the deposition of abnormal aggregates of alpha-synuclein protein in the brain which are in the form of Lewy bodies. Dopaminergic neurons from the SNpc project into a large subcortical structure known as the striatum, and positron emission tomography (PET) studies have demonstrated the dysfunction of the dopaminergic system in the striatum of patients with PD. Furthermore, immunohistochemistry studies of the striatum have identified the degeneration of dopaminergic nerve terminals and inclusions of alpha-synuclein. An aggressive and early onset form of familial PD is caused by the G51D point mutation in alpha-synuclein (G51D/+). Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been used to generate a novel and precise rat model of PD which has the G51D mutation in rat alpha-synuclein. Wild-type (WT) and G51D/+ rats were analysed over the course of ageing (5, 10/11 and 16/17 months of age) using histological experiments and L-3,4-dihydroxy-6-18F-fluorophenylalanine (18FDOPA) PET imaging in order to determine if G51D/+ rats have abnormalities of histological staining and dopaminergic function analogous to those identified in patients with PD. Histological experiments were optimised using WT rat tissue and then used immunohistochemistry for tyrosine hydroxylase (an enzyme involved in the synthesis of dopamine) to evaluate dopamine nerve terminal integrity in the striatum of WT and G51D/+ rats. In addition, immunohistochemistry for alpha-synuclein was used to evaluate staining for alpha-synuclein in cell bodies and the neuropil within the striatum of WT and G51D/+ rats. 18F-DOPA is a well validated PET radiotracer and has been used to investigate dopaminergic function in the striatum of rats. The enzyme aromatic L-amino acid decarboxylase converts 18F-DOPA to 6-18F-fluorodopamine, which is in turn incorporated into presynaptic vesicles, and then released into the synaptic cleft following neuronal activation. PET imaging experiments were first optimised using phantoms and WT rats, then the optimised protocols were applied to studies of WT and G51D/+ rats. Results from tyrosine hydroxylase immunohistochemistry at Bregma 0.00 mm identified a trend for decreased optical density of tyrosine hydroxylase staining in the striatum of 5 month G51D/+ rats compared with age-matched WT controls (p=0.15), and in 17 month G51D/+ rats compared with age-matched WT controls (p=0.10). Semi-quantitative analysis of alpha-synuclein immunohistochemistry indicated an increased abundance of alpha-synuclein positive cell somata in the striatum, and decreased punctate terminal staining in the neuropil of G51D/+ rats compared with age-matched WT rats. 18F-DOPA PET imaging experiments indicated a trend for decreased influx rate constant (Ki) of 18F-DOPA in the striatum of 5 month old G51D/+ rats compared with age-matched WT controls (p=0.08), and a trend for decreased distribution volume ratio (DVR) of 18F-DOPA in the striatum relative to the cerebellum of 16 month old G51D/+ rats when compared with age-matched WT controls (p=0.09). 18F-DOPA PET imaging experiments also identified a trend for a decreased effective distribution volume ratio (EDVR) of 18F-DOPA in the striatum relative to the cerebellum (p=0.09) and in turn indicated increased effective dopamine turnover (EDT) (p=0.13) in the striatum of 16 month old G51D/+ rats compared with age-matched WT rats. Therefore, the results indicated abnormalities of dopaminergic function, as well as tyrosine hydroxylase and alpha-synuclein staining in G51D/+ rats compared with age-matched WT controls, and this appeared to have some features of PD in humans. Indices of EDT indicated compensatory changes in dopaminergic function in the striatum of 16 month old G51D/+ rats compared with age-matched WT rats. Additional compensatory changes in dopaminergic terminal function and tyrosine hydroxylase protein expression may be evident in 11 and 10 month old G51D/+ rats respectively compared with age-matched WT rats. The G51D/+ rat model represents an interesting model for further studies such as the underlying pathophysiology of PD. However, the phenotype observed in G51D/+ rats appeared to be less severe than that which has been observed in humans with G51D type PD.

In certain neurodegenerative diseases such Dementia with Lewy Bodies (DLB), it is hypothesised that misfolded α-synuclein (α-syn) and β-amyloid both contribute to pathology. α-Syn and β-amyloid have been suggested to synergistically promote one another’s accumulation and aggregation, but the mechanisms are unknown. β-Amyloid is generated from β-/γ-secretase-mediated processing of the amyloid precursor protein (APP). This study investigated how α-syn overexpression in cells affects β-amyloid production from APP, using multiplex assays, luciferase reporter assays, and western blotting. Wildtype α-syn expression induces β-amyloid generation from APP in SH-SY5Y human neuroblastoma cells, and similar changes to APP processing occur in another neuronal cell model. Dominant-negative overexpression of α-syn mutants revealed that disrupting the N-terminal domain can increase APP amyloidogenic processing. Secretase enzymes that perform APP processing were next investigated. γ-Secretase activity, measured by a luciferase reporter, was not increased by α-syn overexpression. A higher ratio of β- to α-secretase processing was hypothesised, which led to expression and activity studies of the major β- and α-secretases, BACE1 and ADAM10 respectively. It was shown that the BACE1 protein expression is post-transcriptionally upregulated in α-syn cells, with increased APP cleavage in cells. ADAM10 protein expression is transcriptionally suppressed in wild-type α-syn cells, reducing total levels of catalytically active enzyme. However the change in ADAM10-mediated APP processing may be negligible since, critically, plasma membrane expression of ADAM10 appears to be maintained. To aid understanding of the mechanism that connects α-syn to APP processing, BACE1 expression was used in pharmacological studies of cell stress signalling. This approach revealed that in α-syn cells BACE1 lysosomal and/or proteasomal degradation may be disturbed. Additionally, BACE1 expression is induced by translational de-repression mediated by eIF2α ser-51 phosphorylation, which was increased in α-syn cells. Although preliminary, the data suggests a role for oxidative stress mediating the increased BACE1 expression in wild-type α-syn cells.

The role of RNA processing in the pathogenesis of neurodegenerative diseases is still poorly understood. α-synuclein (SNCA) is a presynaptic neuronal protein known as the major component of Lewy bodies, the pathological hallmark of Parkinson’s disease (PD). Recent evidence suggests a link between the pathogenesis of PD and the expression of SNCA mRNA isoforms with 3’ untranslated region (3’UTR) of different lengths. The purpose of my doctoral studies was the discovery of RNA-binding proteins (RBPs) regulating SNCA at the post-transcriptional level. Using computational and experimental approaches, I identified a number of trans-acting elements that physically interact with SNCA and potentially control its metabolism. I especially focused on the characterization of two RBPs, ELAVL1 and TIAR, and showed their implication in SNCA mRNA stability and translation efficiency. These two factors might play important roles in the maintenance of α-synuclein level and functionality in physiological and pathological conditions.
Se sap ben poc sobre el paper del processament del RNA en la patogènesi de les malalties neurodegeneratives. L’α-sinucleïna (SNCA) és una proteïna neuronal presinàptica i el principal component dels cossos de Lewy, que al seu torn són la troballa patològica característica en la malaltia de Parkinson (MP). Recentment s’ha suggerit un lligam entre la patogènesi de la MP i l’expressió d’isoformes del mRNA de SNCA amb diferents longituds de les regions de 3’ no traduïdes (en anglès conegudes com a untranslated regions UTRs). El propòsit dels meus estudis de doctorat és descobrir les proteïnes que uneixen el RNA de SNCA i el regulen a nivell posttranscripcional. Gràcies a una combinació d’estratègies computacionals i experimentals he identificat elements que interaccionen físicament amb SNCA i potencialment en regulen el metabolisme actuant en trans. M’he centrat en la caracterització de dues proteïnes que uneixen RNA, ELAVL1 i TIAR, i mostro la seva implicació en la regulació de l’estabilitat del mRNA i l’eficiència en la seva traducció. Aquestes dues proteïnes podrien tenir un paper clau en el manteniment dels nivells de α-sinucleïna i la seva funció en condicions fisiològiques i patològiques.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder affecting one million Americans. Despite its social and economic impact, the pathological cascades that lead to neuron dysfunction and degeneration in PD are poorly understood. Endoplasmic reticulum (ER) stress has been implicated as an initiator or contributing factor in neurodegenerative diseases including PD. The ER is an organelle central to protein folding and intracellular Ca2+ homeostasis. Perturbations of these functions result in ER stress and upregulation of ER stress proteins, of which some have been implicated in counteracting ER stress-induced cell death. The mechanisms that lead to ER stress and how ER stress proteins contribute to the degenerative cascades remain unclear but their understanding is critical to devising effective therapies for PD. Both the accumulation of mutant a-synuclein (aSyn), which causes an inherited form of PD, and the inhibition of mitochondrial complex I function by PD-inducing neurotoxin lead to ER stress. The critical involvement of ER stress in experimental models of PD supports its potential relevance to PD pathogenesis and led us to test the hypothesis whether the homocysteine-inducible ER protein (Herp), an ubiquitin-like domain (UBD) containing ER-resident protein, can counteract mutant Alpha Syn- and neurotoxin- induced pathological cascades.
ID: 031001465; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Title from PDF title page (viewed July 10, 2013).; Thesis (Ph.D.)--University of Central Florida, 2011.; Includes bibliographical references.
Ph.D.
Doctorate
Molecular Biology and Microbiology
Medicine
Biomedical Sciences

Thesis (M.A.) PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Parkinson’s Disease (PD) is a devastating progressive neurodegenerative disorder second only to Alzheimer's disease in prevalence. Progression is insidious and PD symptomology manifests when approximately half of the DA neurons projecting from the substantia nigra pars compacta (SNpc) to the striatum are lost. PD is histologically characterized by the presence of intracytoplasmic inclusions primarily composed of hyper-phosphorylated and ubiquinated α-synuclein (SNCA), known as Lewy Bodies. Conserved LB pathology in α-synuclein and LRRK2 mediated disease suggests a common pathological pathway in disease progression. In order to address the potential disease-relevant nexus between these two proteins, we generated transgenic C. elegans lines co-expressing LRRK2 (pan-neuronal) and α-synuclein (dopaminergic neuron specific). We report increased and progressive DA-ergic neuron loss in nematodes co-expressing disease linked mutant LRRK2 and α-synuclein compared to nematode lines expressing only α-synuclein. Also, guided by previous CLR network analysis, we implicated mis-regulation of proteostasis machinery in disease progression by demonstrating differential effects of LRRK2 co-expressed with α-synuclein on macroautophagy in our nematode lines expressing LGG, a marker for autophagic flux. Our studies show overexpression of G2019S LRRK2 inhibits autophagy and accelerates age-related dopaminergic neuron toxicity whereas overexpression of WT LRRK2 does not. Cooverexpression of a-synuclein caused increased inhibition of autophagy and showed an increase in DA-ergic neuron degeneration. Although we have no concrete evidence of interaction, we suggest that LRRK2 demonstrates an agedependent interaction with a-synuclein, which potentiates degeneration of dopaminergic neurons.
2031-01-01

Thesis (M.A.)--Boston University
Mutations in alpha-synuclein and leucine-rich repeat kinase 2 (LRRK2) have been implicated in the cause of Parkinson’s disease (PD). These two proteins have been the targets of a great deal of recent research that has transformed our understanding of this disorder. Recent research using C. elegans as a model species has shown that alpha- synuclein expression and the LRRK2-G2019S mutation potentiate neurodegeneration similar to that seen in cases PD. Further exploration revealed that defects in autophagy of dopaminergic neurons may be the cause for the observed pathology. In the current study, the confirmation of autophagy as a possible cause of pathology due to the expression of alpha-synuclein and the LRRK2-G2019S mutation is completed through the use of electron microscopy. We observed that large vacuoles had formed in the cephalic dopaminergic neurons of alpha-synuclein + LRRK2 transgenic samples not seen in wild-type samples. Further, large morphological changes in the nerve ring area of the transgenic nematodes were also observed that may implicate that alpha- synuclein expression in conjunction with the LRRK2-G2019S mutation may have a widespread effect on many neurons that was not previously expected.

La maladie de Parkinson (MP) est caractérisée par l’agrégation de l’α-synucléine (α-syn) et la dégénérescence sélective des neurones dopaminergiques associée à des défauts locomoteurs. L’étiologie de la MP reste incomplète à ce jour mais ses origines multifactorielles impliquent à la fois des facteurs génétiques et environnementaux. La caractérisation de modèles d’induction mixte de la MP permettrait d’élucider les effets toxiques d’une exposition chronique à un agent chimique sur les mécanismes favorisant l’agrégation de l’α-syn. Dans cette étude, notre objectif était de caractériser les effets d’une exposition chronique au paraquat (PQ) de Drosophiles exprimant une des formes, WT ou mutée [A53T], de l’α-syn humaine dans l’ensemble des neurones.Nous avons mis en évidence que les Drosophiles exprimant l’α-syn [A53T] sont plus sensibles à une exposition chronique au PQ et que cette sensibilité accrue reposerait sur une accumulation d’α-syn et notamment de sa forme pathologique, phosphorylée en sérine 129. La détection de vacuoles d’autophagie et l’accumulation de la protéine Ref(2)-P/p62 reflèteraient un blocage du flux de l’autophagie chez les Drosophiles exprimant l’α-syn [A53T] en condition d’exposition chronique au PQ.La prise en compte les aspects génétiques et environnementaux impliqués dans le développement de la maladie de Parkinson permet de modéliser les cas sporadiques qui représentent près de 90% des patients. Des études plus approfondies utilisant ce modèle mixte permettraient de décrire les mécanismes cellulaires mis en jeu lors d’une exposition chronique à un contaminant de l’environnement et donc d’identifier de nouvelles cibles thérapeutiques potentielles pour lutter contre la maladie de Parkinson
Parkinson's disease (PD) is mainly characterized by the aggregation of α-synuclein (α-syn) into Lewy bodies and the selective degeneration of dopaminergic neurons associated with locomotor defects. The etiology of PD is still incomplete, but its multifactorial origins involve genetic and environmental stress factors. The characterization of animal models expressing human α-syn exposed to pesticides is essential for the identification of the mechanisms underlying the aggregation of α-syn upon chemical exposure. In this study, our goal was to characterize the effects of chronic paraquat (PQ) exposure on Drosophila expressing the WT or mutated [A53T] form of α-syn in all neurons.We showed that the expression of α-syn [A53T] increased the sensitivity to chronic exposure to PQ. In addition, PQ induced α-syn accumulation, especially the pathological form, phosphorylated on serine 129. We also found autophagic vacuoles associated with Ref(2)-P/p62 protein accumulation suggesting an impaired autophagic flux in Drosophila expressing α-syn [A53T] under chronic PQ exposure.By taking into account the genetic and environmental aspects, our approach will allow to identify the main factors responsible for the development of PD in the sporadic cases that represent nearly 90% of patients. Further studies based on this model will allow to explore potential therapies for PD treatment

Parkinson’s disease is an incurable neurodegenerative disease characterized by motor deficits, owing to dopaminergic denervation in the nigrostriatal pathway. The abnormal formation of hallmark Lewy bodies underlies the disease process. The pre-synaptic protein alpha- synuclein (⍺-syn) has prion-like properties arising from its propensity to propagate, seed misfolding, and self-aggregate. Pathogenesis is postulated to arise in olfactory and enteric regions, exploiting connected neuronal pathways to ultimately propagate to the substantia nigra pars compacta. There is little known about the earliest stages of ⍺-syn aggregation and its prion-like propagation mechanisms. Bimolecular fluorescence complementation of ⍺-syn aggregates has allowed us to directly visualize aggregation in transgenic mice and mice transduced with an adeno-associated virus vector. Although our transgenic mice expressed BiSyn in a mosaic fashion that limited utility, we were successful in transducing neurons in the mouse striatum. This work has validated the AAV2/9-CMV-BiSyn approach as groundwork for future systematic studies.

Misfolding and aggregation of alpha-synuclein (a-syn) are associated with a range of neurological disorders, including Parkinson's disease (PD). Fibrillar, insoluble aggregates of a-syn, known as Lewy bodies (LBs) are deposited in the substantia nigra and are a pathological hallmark of PD. a-syn is a natively unstructured protein, co-populating extended and more compact conformational forms under equilibrium. The fine balance of this equilibrium can be shifted due to changes in its environment such as alterations in metal content, ionic strength, free dopamine or others, promoting the assembly of a-syn into toxic conformations. Small, soluble oligomers preceding LB formation are thought to be causative, in vitro, different a-syn oligomers have been produced with alternate biochemical properties. Here the primary objective was to uncover the link between conformation and toxic gain of function by the use of functional assays in combination with ESI-IMS-MS. Epitope mapping procedures indicated that different a-syn oligomers have unique epitope features. Dye binding assays such as ThT and ANS fluorescence inferred that the various oligomer types differ in their amyloidogenicity and hydrophobicity. Furthermore, intracellular aggregation assays, MTT cell proliferation and Ca(ll) influx analysis in SH-SY5Y neuroblastoma cells showed that cellular effects correlated with structural features. ESI-IMS-MS spectra of the different oligomers have been acquired and allowed the conformations of the oligomer subsets to be determined. The oligomers assembled up to a hexameric form with a closed ring-like conformation. These results demonstrated that unique structural features are required for toxicity and that a subset of oligomers with characteristic structures may be pivotal in PD.

Parkinson’s disease (PD) affects 1 in 500 of the UK population. A critical step in disease aetiology is the formation of Lewy bodies (LBs), deposits of aggregated alpha-synuclein (αsyn) as amyloid inclusions, within surviving neurons. It is well established that αsyn within LBs has undergone a variety of post-translational modifications, including S129 phosphorylation, and LBs are known to be rich in metals such as copper. It has however, yet to be established how phosphorylation of αsyn and metal ions influence in the pathology of PD, and if the formation of LBs can be prevented by the use of small molecule inhibitors. Electrospray ionisation-ion mobility spectrometry- mass spectrometry (ESI-IMSMS) was used to investigate the conformational changes to compact states of αsyn known to be linked to amyloid formation, for WT and two αsyn mutants mimicking phosphorylation at S87 and S129, in the presence of copper, and their aggregation was monitored by thioflavin-T assays. Results demonstrate that the conformational state of αsyn can be modulated by interactions with copper, causing an increase in the compact state, leading to an increased rate of aggregation. S129D αsyn showed the highest affinity for copper, and the fastest aggregation rates overall. SH-SY5Y cell culture models were used to investigate intracellular aggregation and phosphorylation state upon copper exposure. Increased copper concentration correlated with increased formation of unmodified and phosphorylated intracellular aggregates, an increase in apoptosis, and decreased cell viability. ESI-IMS-MS demonstrated curcumin and its derivatives were able to disrupt amyloid assembly, via prevention of autofragmentation and dissociation of low-order oligomers, thus preventing the aggregation of αsyn. In SH-SY5Y model used however, curcumins were unable to prevent the metal-induced intracellular aggregation of αsyn. Together, results support the hypothesis that phosphorylation has a key role in PD progression, and demonstrated that modified curcumins may be a potential therapeutic for PD.