Academic literature on the topic 'Alpha synuclein'

AbstractParkinson’s disease is a progressive neurodegenerative disorder characterized by the accumulation of misfolded alpha-synuclein in intraneuronal inclusions known as Lewy bodies and Lewy neurites. Multiple studies strongly implicate the levels of alpha-synuclein as a major risk factor for the onset and progression of Parkinson’s disease. Alpha-synuclein pathology spreads progressively throughout interconnected brain regions but the precise molecular mechanisms underlying the seeding of alpha-synuclein aggregation are still unclear. Here, using stable cell lines expressing alpha-synuclein, we examined the correlation between endogenous alpha-synuclein levels and the seeding propensity by exogenous alpha-synuclein preformed fibrils. We applied biochemical approaches and imaging methods in stable cell lines expressing alpha-synuclein and in primary neurons to determine the impact of alpha-synuclein levels on seeding and aggregation. Our results indicate that the levels of alpha-synuclein define the pattern and severity of aggregation and the extent of p-alpha-synuclein deposition, likely explaining the selective vulnerability of different cell types in synucleinopathies. The elucidation of the cellular processes involved in the pathological aggregation of alpha-synuclein will enable the identification of novel targets and the development of therapeutic strategies for Parkinson’s disease and other synucleinopathies.

Abstract Synucleins (including α-, β- and γ-synucleins) are a group of proteins that are expressed at high levels in the central nervous system. The physiologic function of these proteins is unknown. Alpha-synuclein has been implicated in the pathogenesis of neurodegenerative disorders such as Parkinson's disease and Lewy body dementia, as it is highly expressed in the Lewy bodies from both disorders. The expression of α-synuclein in hematopoietic system has been shown in erythroid precursors and megakaryocytes in bone marrow, as well as erythrocytes and platelets in peripheral blood. Moreover, some studies demonstrated the expression of α-synuclein on peripheral blood mononuclear cells (PBMC), including B and T lymphocytes, NK cells and monocytes; and its expression is shown to be higher in PBMCs of individuals with Parkinson's disease compared to healthy controls. In order to study the role of α-synuclein in development of different hematopoietic elements, we compared bone marrow, peripheral blood and lymphoid organs of age and sex-matched α-synuclein knock-out (KO) mice and wild type (WT) animals of the same genetic background (n=10). Flow cytometric analysis of bone marrow elements did not show differences in the percentages and absolute numbers of erythroid, megakryocytic and myeloid lineages (data not shown). However, differential complete blood cell count (CBC) showed statistically significant decrease in red blood cell (RBC) count, hemoglobin (Hb) and hematocrit (Hct) in KO mice compared to WT mice. No difference was noted in other RBC indices (Table 1). However, platelets were smaller in KO mice as measured by the mean platelet volume (MPV). There was no difference in the number of platelets and white blood cell (WBC) counts. There was a significant reduction in the percentage of circulating lymphocytes, and associated increase in the percentage of neutrophils and monocytes in KO mice compared to WT mice, although the difference in the number of lymphocytes did not reach statistical significance (Table 1). Flow cytometric analysis of T lymphocytes in thymus and peripheral lymphoid organs demonstrated marked defect in development of mature T cells. There was a significant increase in the number of double negative thymocytes in KO mice associated with significant decrease in the number of single positive T cells. Furthermore, splenic CD4+ and CD8+ T cells were markedly decreased in KO mice, indicating that α-synuclein is required for T cell development (Table 2). In summary, our findings indicate an absolute requirement for α-synuclein in development of mature T lymphocytes. The underlying mechanism for this function is subject of future studies. Moreover, while α-synuclein-deficiency does not affect the development of myeloid lineage and platelets, lack of this protein is associated with lower number of erythrocytes, suggesting its role in development and/or survival red blood cells. Disclosures: No relevant conflicts of interest to declare.

Intraneuronal accumulation of misfolded alpha-synuclein in the central and peripheral nervous systems is strongly linked to Parkinson disease (PD) and other related synucleinopathies. In rare inherited forms of PD, point mutations or gene multiplications mediate the formation of alpha-synuclein protein aggregates. However, in most PD cases it is presumed that the combined effects of ageing and environmental factors drive the formation of alpha-synuclein aggregates. Despite advances regarding alpha-synuclein pathobiology, the normal functions of this protein and factors that regulate its expression are not well understood. We discuss a recent study reporting that viral infection induces alpha-synuclein expression in neurons of the gastrointestinal tract. Alpha-synuclein levels increased during norovirus infection in the duodenum of children. In an in vitro paradigm, monomeric and oligomeric alpha-synuclein acted as chemoattractants for neutrophils and monocytes, and promoted the maturation of dendritic cells. This suggests that alpha-synuclein facilitates immune responses to infection. We explore the possibility that intestinal infections, and associated inflammation, place individuals at increased risk of PD by increasing alpha-synuclein levels and promoting the formation of alpha-synuclein aggregates that propagate in a prion-like fashion via the vagal nerve to the brainstem.

Parkinson’s disease (PD) is one of the key neurodegenerative disorders caused by a dopamine deficiency in the striatum due to the death of dopaminergic (DA) neurons of the substantia nigra pars compacta. The initially discovered A53T mutation in the alpha-synuclein gene was linked to the formation of cytotoxic aggregates: Lewy bodies in the DA neurons of PD patients. Further research has contributed to the discovery of beta- and gamma-synucleins, which presumably compensate for the functional loss of either member of the synuclein family. Here, we review research from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity models and various synuclein-knockout animals. We conclude that the differences in the sensitivity of the synuclein-knockout animals compared with the MPTP neurotoxin are due to the ontogenetic selection of early neurons followed by a compensatory effect of beta-synuclein, which optimizes dopamine capture in the synapses. Triple-knockout synuclein studies have confirmed the higher sensitivity of DA neurons to the toxic effects of MPTP. Nonetheless, beta-synuclein could modulate the alpha-synuclein function, preventing its aggregation and loss of function. Overall, the use of knockout animals has helped to solve the riddle of synuclein functions, and these proteins could be promising molecular targets for the development of therapies that are aimed at optimizing the synaptic function of dopaminergic neurons.

Parkinson’s disease (PD) is a progressive health issue and influences an increasingly larger number of people, especially at older ages, affecting the central nervous system (CNS). Alpha-synuclein is a biomarker closely correlated with the CNS and PD. The loss of neuronal cells in the substantia nigra leads to the aggregation of alpha-synuclein in the form of Lewy bodies, and Lewy neuritis is a neuropathological hallmark. The therapeutic approach of PD focuses on alpha-synuclein as an important substrate of PD pathology. So far, research has focused on antialpha-synuclein to minimize the burden of extracellular alpha-synuclein in the brain, and as a consequence, it ameliorates inflammation. Interdigitated electrode (IDE) biosensors are efficient tools for detecting various analytes and were chosen in this study to detect alpha-synuclein on amine-modified surfaces by using antiaptamer-alpha-synuclein as the probe. In addition, a gold nanoparticle-conjugated aptamer was used to enhance the detection limit. The limit of detection for the binding between alpha-synuclein and aptamer was found to be 10 pM. Control experiments were performed with two closely related proteins, amyloid-beta and tau, to reveal the specificity; the results show that the aptamer only recognized alpha-synuclein. The proposed strategy helps to identify the binding of aptamer and alpha-synuclein and provides a possible method to lower alpha-synuclein levels and inflammation in PD patients.

The accumulation of the various products of alpha-synuclein aggregation has been associated with the etiology and pathogenesis of several neurodegenerative conditions, including both familial and sporadic forms of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is now well established that the aggregation and spread of alpha-synuclein aggregation pathology activate numerous pathogenic mechanisms that contribute to neurodegeneration and, ultimately, to disease progression. Therefore, the development of a safe and effective disease-modifying therapy that limits or prevents the accumulation of the toxic intermediate products of alpha-synuclein aggregation and the spread of alpha-synuclein aggregation pathology could provide significant positive clinical outcomes in PD/DLB cohorts. It has been suggested that this goal can be achieved by reducing the intracellular and/or extracellular levels of monomeric and already aggregated alpha-synuclein. The principal aim of this review is to critically evaluate the potential of therapeutic strategies that target the post-transcriptional steps of alpha-synuclein production and immunotherapy-based approaches to alpha-synuclein degradation in PD/DLB patients. Strategies aimed at the downregulation of alpha-synuclein production are at an early preclinical stage of drug development and, although they have shown promise in animal models of alpha-synuclein aggregation, many limitations need to be resolved before in-human clinical trials can be seriously considered. In contrast, many strategies aimed at the degradation of alpha-synuclein using immunotherapeutic approaches are at a more advanced stage of development, with some in-human Phase II clinical trials currently in progress. Translational barriers for both strategies include the limitations of alpha-synuclein aggregation models, poor understanding of the therapeutic window for the alpha-synuclein knockdown, and variability in alpha-synuclein pathology across patient cohorts. Overcoming such barriers should be the main focus of further studies. However, it is already clear that these strategies do have the potential to achieve a disease-modifying effect in PD and DLB.

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.

AbstractAlpha-synuclein (αSyn) is a conformationally flexible protein that is known to be involved in key neuronal biological processes and the pathogenesis of Parkinson’s disease (PD). The aggregation of αSyn in the brain is not only the neuropathological hallmark of PD but also characterizes other primary synucleinopathies, including dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These disorders share common clinical motor symptoms, namely, parkinsonism, which is associated with the loss of nigral dopaminergic neurons. Pathologically the selective neurodegeneration in synucleinopathies is linked to αSyn dysfunction and its abnormal interactions with other proteins. It should be noted that the aggregation of αSyn is not an absolute pathogenic cause for clinically or genetically diagnosed PD, as a minority of patients have substantial loss of nigral dopaminergic neurons in the absence of αSyn pathology. In addition, αSyn pathology is a commonly observed autopsy finding in asymptomatic senescent brains, confirming that αSyn pathology can occur independently of parkinsonism. Presently, there is no causative treatment for synucleinopathies; therefore, targeting αSyn remains the key research focus. Despite the importance of αSyn in the pathology of synucleinopathies, there is no consensus on reliable markers for defining toxic pathogenic αSyn aggregates in postmortem human brain tissue or on any critical αSyn-inducing pathological events. As such, there is an urgent need to establish a panel of antibodies and animal models for studying synucleinopathies. This chapter summarizes the traditional and recently developed methodologies in the field with suggestions on toolkits to assess the role of αSyn pathology.

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

Background: Antibiotics exposure is related to gastrointestinal tract dysbiosis and appearance of systemic repercussions. Due to the correlation between Enteric Nervous System (ENS) and Central Nervous System (CNS), abnormalities in the gut microbiota have been associated with neurological disorders including Parkinson’s Disease (PD). Objectives: Search evidence in the scientific literature relating antibiotic therapy and Parkinson’s disease. Methods: A systematic review has been done using the descriptors “Parkinson’s disease”, “antibiotics” and “gut microbiota” in PubMed’s database. The research was conducted in april 2021, without temporal limitations, in english and portuguese. Results: Studies suggest that PD begins with intestinal inflammation and abnormal alpha-synuclein deposition in the ENS that follows, through nerves, to the CNS. Results show that leaky gut and dysbiosis preceded 5-10 years PD’s initial symptoms, while the intense exposure to antibiotics preceded 10-15 years the diagnostic. On average, PD patients received larger amounts of antibiotics than controls (p=0.021). Dysbiosis post-antibiotics presented reduced diversity of Bacteroidetes, Firmicutes and Prevotellaceae and growthing of Enterobacteriaceae, resulting in higher risk of gastrointestinal infections, higher rates of pro-inflammatory cytokines, increased permeability of gastrointestinal and brain-blood barriers and hyperexpression of the alpha-synuclein protein in the colon. Conclusion: Poorly controlled antibiotic therapy and its subsequent damage to gut microbiota anticipates PD’s early symptoms.