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25 p. : il.-- Bibliogr.: p. 22-25 Introducción. La enfermedad de Parkinson (EP) es una enfermedad neurodegenerativa caracterizada por la denervación de las neuronas dopaminérgicas de la sustancia negra pars compacta. Los tratamientos farmacológicos actuales se basan en el tratamiento sintomático de las manifestaciones motoras, sin embargo, no modifican el curso de la enfermedad. Teniendo en cuenta que los acúmulos de -sinucleína (-sin) están relacionados con la fisiopatología de la enfermedad, podría ser de interés el uso de moléculas que inhiban la progresión de estos acúmulos, o incluso, favorezcan su degradación. Objetivos. Revisar las posibles estrategias terapéuticas dirigidas a la inhibición de los agregados patológicos de -sin mediante la inmunización activa y pasiva, y la mejora en la degradación de estos acúmulos. Métodos. Se llevó a cabo una revisión sistemática cualitativa siguiendo las normas PRISMA de los estudios encontrados en PubMed. Se incluyeron un total de 17 estudios en la revisión: 9 ensayos clínicos y 8 estudios preclínicos. Resultados y conclusiones. Los ensayos preclínicos demuestran una actividad favorable para evitar la acumulación de -sin mediante procesos de inmunización. Al igual que con el nilotinib, que mejora la degradación de los acúmulos de -sin favoreciendo los mecanismos de autofagia. Los ensayos clínicos definen una seguridad y tolerabilidad favorable de fármacos como el prasenizumab (PRX002), cinpanemab (BIIB054) y nilotinib. No obstante, estas estrategias terapéuticas son todavía muy novedosas. Actualmente, existen ensayos clínicos en desarrollo, por tanto, cabe esperar que en un futuro cercano obtengamos información relevante acerca de la relación beneficio/riesgo de estos tipos de tratamiento.

Dissertação de mestrado em Molecular Genetics, The term "aging" refers to the gradual accumulation of changes that take place progressively over time. These changes are either connected with or responsible for the ever-increasing vulnerability to disease and mortality that comes with growing age. The process of aging is responsible for these changes that occur as a result of the passage of time. Notably, cells, in order to maintain their own viability in the face of threats to the integrity of their proteomes, such as those posed by factors like environmental damage or the natural aging process, have evolved very sophisticated systems of adaptability. Thus, aging can be modulated by cell-non-autonomous mechanisms, which comprise a set of pathways that allows for the coordinated response of several cells to a potentially lethal stimulus. A plethora of findings suggests that cells in the vicinity may communicate with one another by releasing and taking up small molecules. However, there are still many unknowns related to the mechanisms that regulate this intercellular communication, it seems that the unconventional autophagy of cells is a key factor behind the release of small transmissible molecules. Synucleinopathies, like Parkinson's disease, is caused by the spread of alpha-synuclein (aSyn) aggregates across neural networks, which disrupts cellular homeostasis. According to research conducted on neurons, aSyn has the capability to behave in a manner similar to that of a prion and to be released into the extracellular space, where it may subsequently be taken up by the cells that are located nearby. Due to conserved cellular processes from yeast to humans, in this thesis, Saccharomyces cerevisiae was used as a cellular model to investigate the secretion of aSyn to the extracellular environment during chronological aging. We have shown that the secretion of aSyn to the extracellular milieu and chronological aging have a strong correlation with the aSyn expression levels. We also evaluated the mechanism that may be responsible for the secretion of aSyn during the chronological lifespan. Based on our findings, we may deduce that inhibition of the biogenesis of MVBs, as well as the ESCRT machinery, can strongly block the secretion of aSyn while they relatively have a small impact on the chronological lifespan of cells. In conclusion, in this thesis, we went through yeast aging, secretion of aSyn with different expression levels, and the mechanism behind it, while we have designed a novel system for further screening of horizontal transmission of the aSyn with the yeast cells., O termo "envelhecimento" refere-se à acumulação gradual de danos que ocorrem progressivamente ao longo do tempo. Estas mudanças estão ligadas ou são responsáveis pela vulnerabilidade, cada vez maior, à doença e à mortalidade que advém da idade. Notavelmente, as células, para manter a sua própria viabilidade face a ameaças à integridade do seu proteoma, dirigidas por diversos fatores, tais como alterações ambientais ou o processo de envelhecimento natural, desenvolveram sistemas de adaptabilidade muito sofisticados. Neste sentido, o envelhecimento pode ser regulado por mecanismos celulares-não autónomos, que envolvem um conjunto de processo que permitem uma resposta coordenada de várias células a um estímulo potencialmente letal. Evidências sugerem que células próximas podem comunicar umas com as outras, através da secreção e captação de pequenas moléculas. Embora existam ainda muitas incógnitas em relação aos mecanismos associados a esta comunicação entre células, é sugerido que a autofagia não convencional é um dos fatores chave que controlam a libertação de moléculas transmissíveis. As sinucleínopatias, como é exemplo a doença de Parkinson, são causadas pela propagação de agregados de alfa-sinucleína (aSyn) através das redes neuronais, perturbando a homeostase celular. De acordo com estudos realizados em neurónios, a aSyn tem a capacidade de se comportar de uma forma semelhante à de um prião e ser libertada no espaço extracelular, onde pode ser posteriormente ser captada pelas células que estão localizadas nas proximidades. Devido à conservação dos processos celulares, desde a levedura até aos humanos, nesta tese, a levedura Saccharomyces cerevisiae foi o modelo celular usado para estudar os mecanismos relacionadas com a secreção de aSyn para o ambiente extracelular, durante o envelhecimento cronológico. Os resultados aqui apresentados mostram que os níveis de expressão de aSyn têm uma forte correlação com a sua secreção para o meio extracelular, bem como com o envelhecimento cronológico. Também avaliámos o mecanismo que pode ser responsável pela secreção de aSyn durante o tempo de vida cronológico. Com base nos nossos resultados, podemos deduzir que a inibição da biogénese dos MVBs, bem como a maquinaria ESCRT podem bloquear a secreção de aSyn e regular o envelhecimento cronológico das células. Em suma, nesta tese, avaliámos o envelhecimento cronológico da levedura, a sua correlação com a secreção de aSyn com diferentes níveis de expressão, e os mecanismos associados a essa mesma secreção, enquanto desenhamos um novo sistema para posteriormente avaliar a transmissão horizontal da aSyn, usando como modelo a levedura., The work presented in this thesis was performed in the Life and Health Sciences Research Institute (ICVS), University of Minho, Financial support was provided by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020.

For the discovery of treatments against synucleinopathies, it is necessary to unravel and fully understand the mechanism of fibrillation of proteins involved. Among them, a-synuclein (aS) plays a key role in the development of these diseases through its aggregation into oligomers found in Lewy bodies. However, its structural disorder as an intrinsically disordered protein (IDP) makes its characterization by experimental techniques arduously difficult. Atomistic simulations aim to provide insights into this blank canvas and, fortunately, some studies have already suggested promising mechanisms. Still, it is urgent to consider the IDP features in simulations, so recently a lot of force fields designed to deal with IDPs have been developed. In this study, we have carried out a total of 12 ms simulations of an aS core fragment using a popular ff14SB AMBER force field and the ff14IDPSFF variation that includes a grid-based energy correction map (CMAP) method. The predicted chemical shifts from the simulations and those measured from the aS protein in the NMR solution indicate that ff14IDPSFF reproduces the experimental data more accurately. Moreover, structural analysis exhibits opposite trends between secondary structure propensities. The ff14SB force field preserves the a-helices found in the micelle-bound aS structure, which is used as an initial conformation, while ff14IDPSFF stands out with increased structural disorder and the formation of b-sheets, which suggests that the IDP-specific force field can capture more suitable conformations representing the possible intermediate states of the fibrillation process.

Treball de fi de grau en Bioinformàtica. Curs 2021-2022 Tutor: Andreu Paytuví Anàlisi del microbioma intestinal amb tècniques d’Intel·ligència Artificial per tal de descobrir biomarcadors de la malaltia de Parkinson a través de diferències entre el microbioma intestinal de subjectes controls i pacients de Parkinson, mitjançant la identificació de tàxons candidats, famílies de gens i vies biològiques per conèixer variables que podrien arribar a ser importants per a la detecció precoç de la malaltia. Análisis del microbioma intestinal con técnicas de Inteligencia Artificial para el decubrimiento de biomarcadores en la enfermedad del Parkinson a través de diferencias entre el microbioma intestinal de sujetos controles y pacientes con Parkinson, mediante la identificación de taxones candidatos, familias de genes y vías biológicas para obtener información sobre variables importantes en la detección temprana de la enfermedad. Analysis of the gut microbiome with Artificial Intelligence techniques in order to discover biomarkers for Parkinson’s disease through differences between the gut microbiome of controls subjects and Parkinson patients, by identifying candidate taxa, gene families and pathways to get insight of some variables that could become important for the early detection of the disease.

Resumen Introducción. El consumo excesivo de alcohol resulta en neuroadaptación, neurodegeneración y expresión diferencial de numerosos genes. Objetivo. Determinar la relación entre la expresión del gen de la alfa sinucleína (SNCA) en sangre, las variantes de nucleótido único (Single Nucleotide Variant, SNV) en su región promotora y el estreñimiento crónico en personas con problemas de consumo de alcohol. Materiales y métodos. La muestra estuvo conformada por 35 controles y 27 casos, seleccionados según el puntaje obtenido con la herramienta AUDIT. En el diagnóstico del estreñimiento se aplicaron los criterios de Roma IV. La extracción de ácidos nucleicos se hizo a partir de sangre periférica y se evaluó la expresión del gen mediante qPCR, la cuantificación proteica por ELISA y la presencia de SNV en la región promotora del gen por la secuenciación de Sanger. Resultados. Se observó sobreexpresión génica relativa de ARNm del gen SNCA en el grupo de casos sin relación con el estreñimiento crónico. Se evidenció un riesgo 4,8 veces mayor de presentar estreñimiento en el grupo de casos. Se encontraron nueve variantes de nucleótido simple en un segmento de la región promotora del gen rica en secuencias reguladoras CpG, con frecuencia similar entre los grupos, y se detectó una variante en la posición -2171 que no se encuentra reportada en GenBank para variantes clínicas y cuyo genotipo A/T se relacionó con el incremento de la expresión del ARNm del SNCA. Conclusión. En personas con problemas de consumo de alcohol se evidenció la sobreexpresión del ARNm de alfa sinucleína, lo cual no se relacionó con el diagnóstico de estreñimiento crónico. Abstract Introduction: Excessive alcohol consumption results in neuroadaptation, neurodegeneration, and differential expression of numerous genes. Objective: To determine the relationship between the expression of the alpha synuclein gene (SNCA) in blood, single nucleotide variant (SNV) in its promoter region, and chronic constipation in people with problems of alcohol consumption. Materials and methods: The sample consisted of 35 controls and 27 cases selected according to the score obtained with the AUDIT tool. For the diagnosis of constipation, the Rome IV criteria were applied. Nucleic acid extraction was performed from peripheral blood and the expression of the gene was evaluated by qPCR, protein quantification by ELISA, and the presence of SNV in the promoter region of the gene by Sanger sequencing. Results: We observed a relative gene overexpression of SNCA mRNA in the case group, which was not related to the diagnosis of chronic constipation. There was 4.8 times greater risk of presenting constipation in the group of cases. Besides, nine single nucleotide variants were found in a segment of the promoter region of the gene rich in CpG regulatory sequences with similar frequency between the groups while a variant was identified in position -2171, which is not reported in GenBank for variants and whose genotype A/T was associated with increased expression of SNCA mRNA. Conclusion: We evidenced an overexpression of alpha synuclein mRNA in people with problems of alcohol consumption that was not related to the diagnosis of chronic constipation.

Tese de Doutoramento em Ciências da Saúde, The presence of aggregates containing alpha-synuclein (a-syn) and selective loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) are the main characteristics of Parkinson’s Disease (PD). The secretome of mesenchymal stromal cells (MSCs) is the product of their secretory activity and is constituted by a panoply of vesicles, soluble proteins, lipids, and nucleic acids. These molecules, with broad and important roles, are essentially growth factors, anti-inflammatory and anti apoptotic factors, able to exert protective and regenerative actions in the brain tissue. Given the complexity of the mechanisms underlying PD pathophysiology and the richness of the secretome, its application in PD has been investigated, and different studies confirmed its therapeutic potential. Despite the weight of a-syn in the progression of PD, few studies have explored the effects of secretome in models that develop a-syn-mediated pathology. This thesis explored the effects of MSCs-derived secretome in Caenorhabditis elegans models that overexpress a-syn. We found that secretome not only reduced a-syn aggregation but also prevented the a-syn-induced DA neuron loss. Bioreactors are essential tools for large-scale production of secretome and they can alter MSCs secretion activity. We used two dynamic culture systems with distinct design to produce secretome. The capacity of the secretomes to induce neurodifferentiation and to protect DA neurons from the effects of a-syn was compared. The secretomes had different impacts in these models, which was consistent with their distinct protein profiles. Nevertheless, we concluded that the dynamic system, as well as other conditions may have contributed to these results. Finally, we attempted to establish in our laboratory an animal model (rat) of PD, based on the viral vector-mediated overexpression of a-syn, and test the secretome with the best outcomes in the previous study. We observed that animals displayed a significant loss of DA neurons of the SNpc and striatal DA terminals. Moreover, these animals had tendentially a lower motor performance. Secretome-treated animals presented an inferior DA neuron loss, reduced levels of a-syn in the SNpc, and a slightly higher motor performance. In summary, this work provided the basis for future studies using secretome in C. elegans models of PD. It provided new clues regarding the effects of different culturing protocols in the therapeutic potential of secretome and it also gathered more evidence regarding the neuroprotective capacity of the secretome from MSCs., A presença de agregados contendo alfa-sinucleína (a-syn) e a perda seletiva de neurónios dopaminérgicos na substantia nigra pars compacta (SNpc) constituem as principais características da doença de Parkinson (PD). O secretoma das células estromais mesenquimatosas (CEMs) é constituído por uma panóplia de vesículas, proteínas solúveis, lípidos e ácidos nucleicos. Estas moléculas, com amplas e importantes funções, são essencialmente fatores de crescimento, fatores anti-inflamatórios e anti-apoptóticos, capazes de exercer ações protetoras e regenerativas no tecido cerebral. Dada a complexidade dos mecanismos subjacentes à fisiopatologia da PD e a riqueza do secretoma, a sua aplicação na PD tem sido investigada e diversos estudos confirmaram o seu potencial terapêutico. Apesar do peso da a-syn na progressão da PD, poucos estudos exploraram os efeitos do secretoma em modelos que desenvolvem a patologia mediada por a-syn. Esta tese explorou os efeitos do secretoma em modelos de Caenorhabditis elegans que sobre-expressam a-syn. O secretoma de CEMs não só reduziu a agregação de a-syn, mas também evitou a perda de neurónios dopaminérgicos induzida por esta proteína. Os biorreatores são ferramentas essenciais para a produção em larga escala de secretoma e podem também alterar a atividade secretora das CEMs. Assim, usámos dois sistemas dinâmicos com design distintos para produzir secretoma e comparámos a capacidade dos secretomas em induzir neurodiferenciação e proteger os neurónios dopaminérgicos dos efeitos da a syn. Os secretomas tiveram impactos diferentes nesses modelos, o que foi consistente com perfis proteicos distintos. No entanto, concluímos que não só o sistema dinâmico, mas também outras condições, poderão ter contribuído para estes resultados. Finalmente, tentámos estabelecer no nosso laboratório um modelo animal (rato) de PD, baseado na sobre-expressão de a-syn através de um vetor viral e testámos neste modelo o secretoma com os melhores resultados no estudo anterior. Os animais apresentaram uma perda significativa de neurónios dopaminérgicos na SNpc e estriado. Além disso, estes animais apresentaram tendencialmente um desempenho motor inferior. Os animais tratados com secretoma apresentaram uma perda neuronal inferior, níveis inferiores de a-syn na SNpc e um desempenho ligeiramente superior. Em resumo, este trabalho criou a base para estudos futuros usando secretoma em modelos de PD de C. elegans. Forneceu novas pistas sobre os efeitos de diferentes protocolos de cultura no potencial terapêutico do secretoma e reuniu mais evidências acerca da capacidade neuroprotetora do secretoma de MSCs., Financial support was provided by grants from the ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI - Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122; by National funds, through the Foundation for Science and Technology (FCT) - project UIDB/50026/2020 and UIDP/50026/2020. CRM acknowledges FCT and Stemmatters, Biotecnologia e Medicina Regenerativa S.A., for the financial support (PD/BDE/127833/2016).

Background. Salivary α-synuclein (aSyn) and its nitrated form, or 3-nitrotyrosine-α-synuclein (3-NT-αSyn), hold promise as biomarkers for idiopathic Parkinson’s disease (IPD). Nitrative stress that is characterized by an excess of 3-nitrotyrosine proteins (3-NT-proteins) has been proposed as a pathogenic mechanism in IPD. The objective is to study the pathological role of native αSyn, 3-NT-αSyn, and 3-NT-proteins in the saliva and submandibulary glands of patients with IPD. Methods. The salivary and serum αSyn and 3-NT-proteins concentration is evaluated with ELISA in patients and controls. Correlations of αSyn and 3-NT-proteins content with clinical features of the disease are examined. Immunohistochemical 3-NT-αSyn expression in submandibulary gland sections is analyzed. Results. (a) Salivary concentration and saliva/serum ratios of native αSyn and 3-NT-proteins are similar in patients and controls, (b) salivary αSyn and 3-NT-proteins do not correlate with any clinical feature, and (c) three patterns of 3-NT-αSyn-positive inclusions are observed on histological sections: rounded “Lewy-type” aggregates of 10–25 µm in diameter, coarse deposits with varied morphology, and spheroid inclusions or bodies of 3–5 µm in diameter. “Lewy-type” and coarse inclusions are observed in the interlobular connective tissue of the gland, and small-sized bodies are located within the cytoplasm of duct cells. “Lewy-type” inclusions are only observed in patients, and the remaining patterns of inclusions are observed in both the patients and controls. Conclusions. The patients’ saliva presents a similar concentration of native αSyn and 3-nitrotyrosine-proteins than that of the controls, and no correlations with clinical features are found. These findings preclude the utility of native αSyn in the saliva as a biomarker, and they indicate the absence of nitrative stress in the saliva and serum of patients. As regards nitrated αSyn, “Lewy-type” inclusions expressing 3-NT-αSyn are observed in the patients, not the controls—a novel finding that suggests that a biopsy of the submandibulary gland, if proven safe, could be a useful technique for diagnosing IPD. Finally, to our knowledge, this is also the first description of 3-NT-αSyn-immunoreactive intracytoplasmic bodies in cells that are located outside the nervous system. These intracytoplasmic bodies are present in duct cells of submandibulary gland sections from all subjects regardless of their pathology, and they can represent an aging or involutional change. Further immunostaining studies with different antibodies and larger samples are needed to validate the data.

Orientador: Matheus de Castro Fonseca Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A doença de Parkinson (DP) é uma doença neurodegenerativa, crônica e progressiva. Em muitos casos de DP, a morte neuronal está relacionada com a presença de agregados intracelulares da proteína alfa-sinucleína (?Syn). A DP pode ser encontrada nas formas familiar e esporádica, no entanto, apesar da DP esporádica ser a mais comum, sua causa ainda não é bem compreendida. Uma hipótese atual indica que devido ao fato de a ?Syn incorretamente estruturada ser encontrada no sistema nervoso entérico (SNE) antes de surgir no cérebro, a DP esporádica pode se originar a partir do intestino e progredir para o sistema nervoso central (SNC). Em adição, recentemente foi descoberto que células enteroendócrinas (CEEs) presentes no epitélio intestinal possuem propriedades semelhantes a neurônios, se conectam com os neurônios do SNE e expressam a ?Syn. Essas células estão voltadas para a luz do intestino e em constante interação com o microbioma intestinal, sujeitas, portanto, à modulação influenciada por este microambiente intestinal. Reunidas, estas características fazem das CEEs uma possível peça-chave para o estudo sobre a origem e evolução da DP esporádica. Assim, o objetivo deste trabalho é investigar os mecanismos moleculares e celulares que podem estar envolvidos na translocação da ?Syn entre as CEEs e as células neuronais. Para isso, ?Syn monomérica foi produzida, purificada, submetida ao processo de formação de fibrilas pré-formadas (PFFs) e devidamente caracterizadas. Em seguida, avaliamos se as PFFs de ?Syn podem ser internalizadas por organoides cerebrais, pelo epitélio intestinal, por CEEs e por células neuronais, e se núcleos de agregação de ?Syn fibrilar podem iniciar a agregação da ?Syn endógena. Buscamos investigar quais os mecanismos envolvidos no processo utilizando as sondas vitais FM1-43, Fluo-4/AM e live imaging. Mecanismos de transferência de PFFs de ?Syn entre as CEEs e neuronais foram estudados por meio de ensaios de co-culturas na presença ou ausência de contato celular físico. Nossos resultados mostram que obtivemos sucesso na produção da ?Syn monomérica e fibrilar, esta última apresentando características de polidispersão e grande raio hidrodinâmico. As PFFs de ?Syn são eficientemente internalizados por organoides cerebrais e diretamente pelo epitélio intestinal de camundongos, o mesmo sendo observado em CEEs e neuronais isoladas e transfectadas com ?Syn-GFP. A internalização de PFFs de aSyn levou a agregação da ?Syn endógena. PFFs de ?Syn iniciam uma resposta intracelular de Ca2+ e concomitante evento de endocitose. Por último, caracterizamos a propagação de PFFs de ?Syn de células enteroendócrinas para neuronais, mostrando que este processo é dependente do contato físico célula a célula e de Rab35 GTPase. Estes insights mecanísticos da transferência de ?Syn, sugerem fortemente a conexão intestino-cérebro como uma possível rota de origem da DP esporádica Abstract: Parkinson's disease (PD) is a chronic, progressive neurodegenerative disease. PD can be found in familial and sporadic forms, and although sporadic PD is the most common, its cause is still not well understood. In many cases, neuronal death is related to the presence of intracellular aggregates of the protein alpha-synuclein (?Syn). A current hypothesis indicates that since incorrectly structured ?Syn is found in the enteric nervous system (ENS) before it appears in the brain, sporadic PD may originate in the gut and progress to the central nervous system (CNS). In addition, it has been recently discovered that enteroendocrine cells (EECs) present in the intestinal epithelium possess neuron-like properties, connect with enteric nerves and express ?Syn. These cells face the gut lumen and are in constant interaction with the gut microbiome, being influenced by these organisms, and therefore, being modulated by the gut microenvironment. Taken together, these features make EECs a key player in the study of the origin and evolution of sporadic PD. Thus, the aim of this work is to investigate the molecular and cellular mechanisms that may be involved in the translocation of ?Syn between EECs and neuronal cells. To this end, ?Syn was produced, purified, subjected to the process of fibril formation (PFFs) and properly characterized. We then evaluated whether ?Syn PFFs can be internalized by brain organoids, the intestinal epithelium, EECs and neuronal cells and, whether these ?Syn seeds can initiate aggregation of endogenous ?Syn and sought to investigate what mechanisms are involved in the process using the vital probes FM1-43, Fluo-4/AM and live imaging. Mechanisms of PFFs transfer of ?Syn between EECs and neuronal cells were studied using co-culture assays in the presence or absence of physic cell-cell contact. We successfully produced monomeric and fibrillar forms of ?Syn, the latter exhibiting polydispersity characteristics and large hydrodynamic radius. We demonstrate that ?Syn PFFs are efficiently internalized by brain organoids and directly by mouse intestinal epithelium, the same being observed in EECs and neuronal cells isolated and transfected with ?Syn-GFP. Interestingly, internalization of aSyn PFFs led to aggregation of endogenous ?Syn. In addition, ?Syn PFFs initiate an intracellular Ca2+ response and concomitant endocytosis event. Lastly, we characterized the spread of ?Syn PFFs from enteroendocrine to neuronal cells showing that this process is dependent on physical cell-to-cell contact and on Rab35 GTPase. Thus, our results provide mechanistic insights into ?Syn transfer, strongly suggesting the gut-brain connection as a possible route of origin of sporadic PD Doutorado Biologia Celular Doutora em Biologia Celular e Estrutural CNPQ 140502/2020-3 CAPES 001

Orientador: Matheus de Castro Fonseca Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: A Doença de Parkinson (DP) é a segunda doença neurodegenerativa mais comum atualmente, atingindo cerca de 10 milhões de pessoas no mundo. Compromete o sistema nervoso central de forma crônica e progressiva, causando danos e morte dos neurônios dopaminérgicos do mesencéfalo, especificamente da substância negra, acarretando diminuição de dopamina no organismo. O indivíduo portador dessa deficiência apresenta como principais sintomas a lentidão motora, rigidez nas articulações, desequilíbrio e tremores involuntários durante repouso. A principal hipótese para a morte dos neurônios é a presença de agregados proteicos denominados Corpos de Lewy (LB’s) compostos principalmente pela alfa-sinucleína (alfa-sin). Essa proteína está intimamente relacionada com mecanismos de transporte de vesícula nas células e se concentra principalmente nos terminais pré-sinápticos dos neurônios, uma região submetida a altos níveis e flutuações de cálcio (Ca2+). Além disso, proteínas da família Rab GTPases (Rab’s) tem relação intima com a alfa-sin, com destaque para a Rab8a. Estudos recentes demonstram que a desregulação da homeostase de Ca2+ em neurônios está intimamente relacionada com o desenvolvimento de doenças neurodegenerativas como a DP, mas os mecanismos envolvidos nesse processo ainda não foram elucidados. Neste trabalho, descrevemos que a perda da homeostase de Ca2+ em células neuronais SH-SY5Y promove a agregação da alfa-sin e faz com que a sua interação com a Rab8a seja intensificada. Além disso, estudos biofísicos in vitro utilizando a alfa-sin recombinante mostraram alterações estruturais da proteína de maneira dependente de sua concentração no meio, mas independente de sua interação com diferentes concentrações de Ca2+. Experimentos com a Rab8a recombinante in vitro, indicam que a interação entre alfa-sin e Rab8a ocorre em condições basais e é intensificada na presença de Ca2+, processo análogo ao observado no interior das células. Assim, os dados obtidos mostram relação direta do aumento de Ca2+ e o mau funcionamento de ambas as proteínas, indicando ser um dos possíveis mecanismos responsáveis para o desenvolvimento de DP Abstract: Parkinson's disease (PD) is the second most common neurodegenerative disease today, affecting about 10 million people worldwide. It affects the central nervous system in a chronic and progressive way, causing damage and death to the dopaminergic neurons of the midbrain, specifically the substantia nigra, leading to a huge decrease in dopamine in the body. The individual with this deficiency presents as main symptoms motor slowness, joint stiffness, unbalance, and involuntary tremors during rest. The main hypothesis for the death of neurons is the presence of protein aggregates called Lewy bodies (LB's), mainly composed of alpha-synuclein (alpha-sin). This protein is closely related to vesicle transport mechanisms in cells and is mainly concentrated in the presynaptic terminals of neurons, a region subjected to high levels and fluctuations of calcium (Ca2+). Moreover, the Rab GTPases proteins family (Rab's) are closely related to alpha-sin, especially Rab8a. Recent studies show that the dysregulation of Ca2+ homeostasis in neurons is closely related to the development of neurodegenerative diseases such as PD, but the mechanisms involved in this process have not yet been elucidated. In this work, we describe that disruption of Ca2+ homeostasis in SH-SY5Y neuronal cells promotes aggregation of alpha-sin and causes its interaction with Rab8a to be intensified. Furthermore, in vitro biophysical studies using recombinant alpha-sin showed structural changes of the protein in a manner dependent on its concentration in the medium but independent of its interaction with different Ca2+ concentrations. Experiments with recombinant Rab8a in vitro indicate that the interaction between alpha-sin and Rab8a occurs under basal conditions and is intensified in the presence of Ca2+, a process analogous to that observed inside the cells. Thus, the data obtained show a direct relationship between increased Ca2+ and the malfunction of both proteins, and this is one of the possible mechanisms responsible for the development of PD Mestrado Biologia Celular Mestre em Biologia Celular e Estrutural CNPQ 130646/2020-2 CAPES 001

Dissertação de Mestrado em Bioquímica apresentada à Faculdade de Ciências e Tecnologia Alfa-sinucleína (a-sin) é uma proteína envolvida em casos esporádicos e alguns familiares da doença de Parkinson (DP), uma doença neurodegenerativa de movimento relacionada com a idade que afeta principalmente os neurónios dopaminérgicos da porção compacta da substância nigra e, progressivamente, todo o córtex. Em sinucleinopatias relacionadas com DP, é perturbada a homeostase de cálcio intracelular, aumentado os níveis de espécies reativas de oxigénio e danificando os sistemas de degradação de proteínas como o sistema da ubiquitina-proteassoma e a via da autofagia-lisossoma. Disfunção mitocondrial já foi amplamente descrita em contexto de PD, incluindo a inibição do complexo I, depleção de energia e aumento dos níveis de espécies reativas de oxigénio mitocondrial, que em conjunto agravam os processos citotóxicos. Adicionalmente, os agregados de a-sin parecem inibir a mitofagia, um processo específico de autofagia que degrada mitocôndrias danificas, fazendo com que estas se mantenham na célula. Tendo em conta que as características da DP podem surgir do acumular de a-sin,WT o principal objetivo deste estudo foi definir a citotoxicidade de eventos ligados à desregulação da degradação de proteínas, principalmente a danificação via da autofagia-lisossoma, e da mitofagia em células de neuroblastoma humano SHSY-5Y com um sistema Tet-Off condicionado. Aqui, é mostrado que a sobre-expressão de a-sin WT causa um aumento nos níveis de espécies reativas de oxigénio e alterações nos níveis de proteínas autofágicas que impede a a-sin WT de ser degradada. Além disso, quando a a-sin WT sobre-expressada colocaliza com a mitocondria, o potencial da membrana mitocondrial é reduzido e a morfologia mitocondrial alterada, o que pode confirmar a degredação seletiva mitocondrial através da via da autofagia-lisssoma/via da mitofagia. Compreender as mudanças na autofagia/macroautofagia e na mitofagia associadas a níveis aumentados de a-sin WT pode ajudar a identificar estratégias neuroprotetoras seletivas destinadas a atenuar a progressão da DP. Alpha-synuclein (a-Syn) is a protein involved in both sporadic and some familial forms of Parkinson’s disease (PD), an age-related neurodegenerative movement disorders that mainly affects dopaminergic neurons in the substantia nigra pars compacta and progressively the whole cortex. In PD-related synucleopathies, the protein forms oligomers that are toxic to the cell, disrupting intracellular calcium homeostasis, increasing the levels of reactive species of oxygen (ROS) and causing the impairment of proteastase mechanisms, such as the ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP). Mitochondrial dysfunction has been also largely described in PD, including complex I inhibition, energy depletion and increased mitochondrial ROS levels, which together exacerbate cytotoxic processes. Additionally, a-syn aggregates seem to abrogate mitophagy, a selective autophagy process that degrades damaged mitochondria, thus maintaining damaged mitochondria in cells. Considering that PD features may arise from accumulated levels of WT a-syn, the main goal of this study was to define the cytotoxic events linked to deregulated proteastase, namely dysfunctional ALP, and mitophagy in human neuroblastoma catecholaminergic Tet-Off conditional SH-SY5Y cells. Here we show that WT a-syn overexpression causes increase cellular ROS levels and alterations in autophagic protein levels that preclude WT a-syn from being degraded. Furthermore, when overexpressed WT a-syn colocalizes with mitochondria leading to decreased mitochondrial membrane potential and altered morphology, which may endorse mitochondrial selective degradation through ALS/mitophagy pathways. Understanding the changes in autophagy/macroautophagy and mitophagy linked to increased levels of WT a-syn may help to identify selective neuroprotective strategies aimed to alleviate PD progression.

A doença de Parkinson (DP) apresenta uma elevada prevalência entre as doenças neurodegenerativas, acarretando um impacto clínico muito significativo nos pacientes, familiares e cuidadores, especialmente através dos seus efeitos degenerativos progressivos na mobilidade do doente. As evidências científicas apontam para a centralidade da alfa sinucleína, uma proteína intrinsecamente desordenada no citosol que é tida como fundamental no desenvolvimento da doença. A etiologia da DP é variada e, consoante o fator desencadeante da patologia, podem ocorrer várias alterações nas vias fisiológicas e na alfa-sinucleína monomérica. Alterações na proteína podem levar a danos nas vias celulares, bem como danos nestas vias podem provocar alterações na proteína. Em última instância ocorre morte neuronal, e em neurónios resilientes são encontrados corpos de Lewy compostos maioritariamente por alfa-sinucleína. Estes corpos são formados porque numa fase inicial ocorrem alterações na homeostase celular que induzem perturbações nos equilíbrios entre as diferentes formas de alfa sinucleína, originando a formação de oligómeros e fibrilas tóxicas. Além dos danos que causam sobre as vias celulares, estas espécies têm a capacidade de se disseminarem de forma semelhante a um prião, interferindo com a homeostase de células vizinhas. As terapias atualmente utilizadas em quadros clínicos de DP apenas controlam sintomas e não inibem a progressão da doença, pelo que o estudo de novas estratégias e fármacos que impeçam essa progressão são de extrema relevância. A presente dissertação centra-se na análise das formas tóxicas da alfa-sinucleína e dos seus mecanismos de toxicidade, abordando estudos conducentes a novas estratégias que atuem sobre esta proteína. De entre as estratégias descritas na literatura, as mais promissoras envolvem a diminuição da produção da alfa-sinucleína, a inibição da sua agregação, a potenciação da autofagia, a redução de formas extracelulares tóxicas bem como inibição da captação das mesmas por células vizinhas. Parkinson’s disease (PD) presents a high prevalence among the neurodegenerative diseases, resulting on a significant clinical impact on patients, their family and care takers, especially through its progressive degenerative effects on patient’s mobility. Scientific evidence points to the centrality of alpha-synuclein, an intrinsically disordered protein in the cytosol known to be crucial for the development of the disease. The etiology of the PD varies, largely depending on the triggering factor of the pathology. Several changes to the physiological pathways and in the monomeric alpha-synuclein may occur. Changes to the protein can lead to damage on the cellular pathways as well as damages in the physiological pathways may alter the protein. At last, neuronal death occurs and Lewy bodies composed mainly of alpha-synuclein are found in resilient neurons. These bodies are formed because in an initial phase changes in cell homeostasis occur, causing disturbances in the balance between the different forms of alpha synuclein which lead to formation of toxic oligomers and fibrils. Aside from the damages caused on cellular pathways, these species have de capacity to spread like a prion, interfering with the homeostasis of neighboring cells. The therapies currently available for PD only control symptoms, not slowing the progression of the disease. The study of new strategies that prevent this progression are thus extremely important. This dissertation focuses on the analysis of toxic forms of alpha-synuclein and their mechanisms of toxicity, together with recent studies described in the literature aiming at the development of new strategies targeting this protein. The most promising approaches are related to reducing alpha-synuclein production, inhibiting its aggregation, potentiating autophagy, reducing toxic extracellular forms as well as inhibiting their uptake by neighboring cells.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Our study aimed to analyze the expression and protein content of four genes (ATF4, MSN, PBXIP1 and SNCA) by qPCR and Western Blotting in individuals with sickle cell anemia treated and not treated with hydroxyurea (HU) in order to establish associations with pathophysiology sickle cell anemia and, mainly, with HU treatment. To prove the results, analyzes were also performed with K562 cells culture stimulated for erythroid differentiation in the presence and absence of HU. Our results show decrease in the expression of MSN in individuals with the disease, which can be justified by the increase in cell adhesion in this group. The data obtained also suggest that treatment with HU induces the expression of PBXIPI and inhibits SNCA. The PBXIPI activation would assist in stimulating the differentiation and proliferation of hematopoietic cells, while the decrease in the content of SNCA may be associated with the reduction of inflammatory processes, which is an effect of great importance in worsening the disease. For the ATF4 gene, associated with oxidative stress and neovascularization, we expected that treatment with hydroxyurea would lead to a reduction in expression, however, we did not obtain a statistically significant difference in the comparison between groups. Finally, we suggest that the increase in oxidative stress by adding H2O2 to the culture medium also influences the expression of our genes and affects the potential effect of hydroxyurea. Nosso estudo se propôs a analisar a expressão e conteúdo proteico de quatro genes (ATF4, MSN, PBXIP1 e SNCA) por qPCR e Western Blotting em indivíduos com anemia falciforme tratados e não tratados com hidroxiureia (HU) a fim de estabelecer associações com a fisiopatologia da anemia falciforme e, principalmente, com o tratamento com HU. Para comprovação dos resultados, as análises também foram realizadas com células K562 estimuladas para diferenciação eritroide na presença e ausência de HU. Nossos resultados mostram uma diminuição da expressão de MSN nos indivíduos com a doença, o que pode ser justificado pelo aumento de adesão celular neste grupo. Os dados obtidos sugerem também que o tratamento com HU induz a expressão de PBXIPI e inibe a de SNCA. A ativação de PBXIPI auxiliaria no estímulo da diferenciação e proliferação de células hematopoiéticas, enquanto que a diminuição no conteúdo de SNCA pode estar associada à redução dos processos inflamatórios, que é um efeito de grande importância no agravamento da doença. Para o gene ATF4, associado ao estresse oxidativo e neovascularização, esperávamos que o tratamento com hidroxiureia levasse a uma redução da expressão, porém, não obtivemos diferença estatística significativa na comparação entre os grupos. Por fim, sugerimos que o aumento do estresse oxidativo pela adição de H2O2 ao meio de cultura também influencia a expressão de nossos genes e afeta o potencial efeito da hidroxiureia. CNPq: 130497/2018-5

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES PARTICIPATION OF NORADRENERGIC RECEPTORS ON MOTOR AND NEUROCHEMICAL CHANGES IN AN EXPERIMENTAL PARKINSONISM MODEL Introduction: Parkinson's disease (PD) is a multifactorial disease with no defined etiology and pathophysiology characterized by progressive death of dopaminergic neurons. However, evidence shows that noradrenergic dysfunction is associated with the progression of PD. Besides, epidemiological studies show that the use of β2-adrenergic agonists reduces the risk of PD. Therefore, this study aimed to evaluate the participation of noradrenergic receptors in response to the monoaminergic deficit induced by reserpine. Method: Wistar rats (6- to 8- month-old) were used. The animals were divided into six groups: control group (CTL); reserpine group (RES); salbutamol group (SALB); reserpine-salbutamol group (RES-SALB); propranolol group (PRO); and reserpine-propranolol group (RES-PRO). All animals received 12 subcutaneous injections of RES solution (0.1 mg/kg) or vehicle (CTL) throughout 25 days, one every 48 hours. After the 16th day (8th injection of RES), all animals were treated intraperitoneally with SALB (5 mg/kg), PRO (20 mg/kg) or vehicle for 8 days, one every 24 hours. The animals were submitted to behavioral tests of catalepsy I (performed every 48 hours, throughout the experiment), catalepsy II (performed on the 16th day - 30, 60, 120 minutes, 24 and 48 hours after the 1st injection of SALB and PRO), open field (performed on the 16th day, 60 minutes after the 1st injection of SALB and PRO) and oral movements (performed on the 16th day, 120 minutes after the 1st injection of SALB and PRO, on the18th and the 24th day). Forty-eight hours after the last injection of RES, the animals were anesthetized, perfused and the brains were submitted to immunohistochemical analysis of tyrosine hydroxylase (TH), dopamine β-hydroxylase (DβH) and α-synuclein (α-syn). Results: It was observed that a single injection of SALB and PRO attenuated the increase of oral movements such as vacuum chewing and tongue protrusion, induced by RES. On the other hand, repeated administration of SALB progressively reduced the time of catalepsy; and PRO attenuated the increase of oral movements (vacuum chewing and oral tremor) induced by RES. Regarding immunohistochemical analysis, it was observed that both SALB and PRO protected against the reduction of TH immunoreactivity in the substantia nigra pars compacta (SNpc), ventral tegmental area (VTA) and striatum, as well as for DβH in the locus coeruleus (LC); and against the increase of α-syn immunoreactivity in the substantia nigra pars reticulata (SNr), striatum, ventral subventricular area (VSA), CA1, CA3, dentate gyrus (DG) and medial prefrontal cortex (mPFC) caused by RES. Conclusion: Our results suggest that the modulation of RAβ promotes a neuroprotective effect on motor and neurochemical changes in the experimental model of parkinsonism induced by RES since an improvement in motor condition and an increase in TH and DβH immunoreactivity and a reduction of α-syn immunoreactivity in the experimental model of parkinsonism were observed. PARTICIPAÇÃO DOS RECEPTORES NORADRENÉRGICOS SOBRE AS ALTERAÇÕES MOTORAS E NEUROQUÍMICAS EM UM MODELO EXPERIMENTAL DE PARKINSONISMO Introdução: A doença de Parkinson (DP) é uma doença multifatorial sem etiologia definida e fisiopatologicamente caracterizada por morte progressiva de neurônios dopaminérgicos. No entanto, indícios mostram que disfunção noradrenérgica está associada a progressão da DP. Além disso, estudos epidemiológicos mostram que o uso de agonistas adrenérgicos β2 reduz o risco para DP. Assim, o objetivo deste estudo foi avaliar a participação dos receptores noradrenérgicos frende aos déficits monoaminérgicos induzido por reserpina. Método: Foram utilizados ratos Wistar de 6 a 8 meses de idade. Os animais foram divididos em seis grupos: grupo controle (CTR); grupo reserpina (RES); grupo salbutamol (SALB); grupo reserpinasalbutamol (RES-SALB); grupo propranolol (PRO); e grupo reserpina-propranolol (RESPRO). Todos os animais receberam 12 injeções por via s.c. da solução RES (0,1 mg/kg) ou veículo (CTR) administradas durante 25 dias, uma a cada 48 horas. Após o 16º (8ªinjeção de RES), todos os animais foram tratados com SALB (5 mg/kg), PRO (20 mg/kg) ou veículo por via i.p., durante 8 dias, uma a cada 24 horas. Os animais foram submetidos aos testes comportamentais de catalepsia I (realizada a cada 48 horas, ao longo de todo experimento), catalepsia II (realizada no 16º dia, 30, 60, 120 minutos, 24 e 48 horas após a 1ª injeção de SALB e PRO), campo aberto (realizado no 16º dia, 60 minutos após a 1ª injeção de SALB e PRO) e movimentos orais (MOFS) (realizado no 16º dia, 120 minutos após a 1ª injeção de SALB e PRO, 18º e 24º dia) e 48 horas após a última injeção de RES, os animais foram anestesiados, perfundidos e os encéfalos submetidos a imuno-histoquímica para tirosina hidroxilase (TH), dopamina β-hidroxilase (DβH) e α-sinucleína (α-syn). Resultados: Observou-se que uma única injeção de SALB e PRO atenuou o aumento nos MOFS, mastigação no vácuo e protusão de língua, induzido por RES. Enquanto a administração repetida de SALB reduziu progressivamente o tempo de catalepsia e PRO atenuou o aumento de MOFS, mastigação no vácuo e tremor oral, induzido por RES. Em relação a expressão imunorreativa, observou-se que tanto SALB quanto PRO protegeu contra a redução da imunorreatividade para TH na substância negra parte compacta (SNpc), área tegmentar ventral (VTA - ventral tegmental area) e estriado, bem como para DβH no locus coeruleus (LC) e contra o aumento da imunorreatividade para αsyn na substância negra reticulada (SNr), estriado, área subventricular ventral (ASV), CA1, CA3, giro denteado (GD e córtex pré-frontal medial (CPFm) provocado por RES. Conclusão: Nossos resultados sugerem que a modulação dos RAβ promove um efeito neuroprotetor sobre as alterações motoras e neuroquímicas do modelo experimental de parkinsonismo induzido por RES, uma vez que foi observada uma melhora na condição motora e aumento na marcação de TH e DβH e redução de α-syn no modelo experimental de parkinsonismo. São Cristóvão, SE

Background: α-Synuclein is a small soluble protein, whose physiological function in the healthy brain is poorly understood. Intracellular inclusions of α-synuclein, referred to as Lewy bodies (LBs), are pathological hallmarks of α- synucleinopathies, such as Parkinson’s disease (PD) or dementia with Lewy bodies (DLB). Main body: Understanding of the molecular basis as well as the factors or conditions promoting α-synuclein misfolding and aggregation is an important step towards the comprehension of pathological mechanism of α- synucleinopathies and for the development of efficient therapeutic strategies. Based on the conversion and aggregation mechanism of α-synuclein, novel diagnostic tests, such as protein misfolding seeded conversion assays, e.g. the real-time quaking-induced conversion (RT-QuIC), had been developed. In diagnostics, α-synuclein RT-QuIC exhibits a specificity between 82 and 100% while the sensitivity varies between 70 and 100% among different laboratories. In addition, the α-synuclein RT-QuIC can be used to study the α-synuclein-seeding-characteristics of different α-synucleinopathies and to differentiate between DLB and PD. Conclusion: The variable diagnostic accuracy of current α-synuclein RT-QuIC occurs due to different protocols, cohorts and material etc.. An impact of micro-environmental factors on the α-synuclein aggregation and conversion process and the occurrence and detection of differential misfolded α-synuclein types or strains might underpin the clinical heterogeneity of α-synucleinopathies.

Tese de mestrado, Bioquímica (Bioquímica Médica) Universidade de Lisboa, Faculdade de Ciências, 2020 Submitted by Teresa Boa (tdboa@fc.ul.pt) on 2021-05-11T17:52:00Z No. of bitstreams: 1 ulfc126263_tm_Ana_Rita_Marçal.pdf: 24634959 bytes, checksum: bcd387b8f0ec9d6b97c0c06993f3612d (MD5) Made available in DSpace on 2021-05-11T17:52:11Z (GMT). No. of bitstreams: 1 ulfc126263_tm_Ana_Rita_Marçal.pdf: 24634959 bytes, checksum: bcd387b8f0ec9d6b97c0c06993f3612d (MD5) Previous issue date: 2020

Tese de mestrado em Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2020 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2021-06-07T16:44:05Z No. of bitstreams: 1 ulfc126405_tm_Luís_Palmares.pdf: 1558059 bytes, checksum: 96f78bcda3a7e2b8b9cdae2ab3f47132 (MD5) Made available in DSpace on 2021-06-07T16:44:14Z (GMT). No. of bitstreams: 1 ulfc126405_tm_Luís_Palmares.pdf: 1558059 bytes, checksum: 96f78bcda3a7e2b8b9cdae2ab3f47132 (MD5) Previous issue date: 2020

Alpha-synuclein (aSyn) is a central player in Parkinson’s disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.

Several studies have indicated that certain misfolded amyloids composed of tau, β-amyloid or α-synuclein can be transferred from cell to cell, suggesting the contribution of mechanisms reminiscent of those by which infective prions spread through the brain. This process of a 'prion-like' spreading between cells is also relevant as a novel putative therapeutic target that could block the spreading of proteinaceous aggregates throughout the brain which may underlie the progressive nature of neurodegenerative diseases. The relevance of β-amyloid oligomers and cellular prion protein (PrPC) binding has been a focus of interest in Alzheimer's disease (AD). At the molecular level, β-amyloid/PrPC interaction takes place in two differently charged clusters of PrPC. In addition to β-amyloid, participation of PrPC in α-synuclein binding and brain spreading also appears to be relevant in α-synucleopathies. This review summarizes current knowledge about PrPC as a putative receptor for amyloid proteins and the physiological consequences of these interactions.

Background Leucine‐rich kinase 2 (LRRK2)‐linked Parkinson's disease (PD) is clinically indistinguishable from idiopathic PD (IPD). A pleiotropic neuropathology has been recognized but the majority of studies in LRRK2 p.G2019S patients reveal Lewy‐type synucleinopathy as its principal histological substrate. To date no in vivo biomarkers of synucleinopathy have been found in LRRK2 mutation carriers. Objectives We used real‐time quaking‐induced conversion (RT‐QuIC) technique to assess the presence of alpha‐synuclein (a‐syn) aggregates in cerebrospinal fluid (CSF) of LRRK2 p.G2019S carriers. Methods CSF samples of 51 subjects were analyzed: 15 LRRK2 p.G2019S PD, 10 IPD, 16 LRRK2 p.G2019S nonmanifesting carriers (NMC) and 10 healthy controls. The presence of parkinsonism and prodromal symptoms was assessed in all study subjects. Results Forty percent (n = 6) LRRK2‐PD, and 18.8% (n = 3) LRRK2‐NMC had a positive a‐syn RT‐QuIC response. RT‐QuIC detected IPD with 90% sensitivity and 80% specificity. No clinical differences were detected between LRRK2‐PD patients with positive and negative RT‐QuIC. A positive RT‐QuIC result in LRRK2‐NMC occurred in a higher proportion of subjects meeting the Movement Disorder Society research criteria for prodromal PD. Interpretation RT‐QuIC detects a‐syn aggregation in CSF in a significant number of patients with LRRK2‐PD, but less frequently than in IPD. A small percentage of LRRK2‐NMC tested also positive. If appropriately validated in long‐term studies with large number of mutation carriers, and hopefully, postmortem or in vivo confirmation of histopathology, RT‐QuIC could contribute to the selection of candidates to receive disease modifying drugs, in particular treatments targeting a‐syn deposition.

Las enfermedades neurodegenerativas son procesos crónicos, progresivos y complejos caracterizadas por la pérdida selectiva de neuronas en los sistemas motor, sensorial y cognitivo. Estas patologías, que actualmente afectan a más de 42 millones de personas en el mundo, están asociadas a la acumulación anormal de agregados amiloides de proteínas específicas en el tejido nervioso. La agregación de α-sinucleína en neuronas dopaminérgicas es la evidencia histopatológica y el principio etiopatológico en la enfermedad de Parkinson (EP) donde las especies oligoméricas de esta proteína son responsables de la muerte neuronal y de la diseminación de la patología. Por otro lado, las especies fibrilares resultantes del proceso de agregación están relacionadas con el inicio y/o desarrollo del proceso neuroinflamatorio característico de la EP. Si bien numerosas moléculas demostraron ser eficientes inhibidores de la agregación amiloide de α-sinucleína in vitro, no han logrado proporcionar una neuroprotección efectiva in vivo y actualmente no existen fármacos capaces de detener y/o revertir el daño neuronal. Estas altas tasas de fracasos son debidas a la toxicidad o incapacidad de atravesar la barrera hematoencefálica de la mayoría de las moléculas estudiadas. En este contexto, el reposicionamiento de drogas ya existentes en la farmacopea sobre nuevos blancos terapéuticos es una estrategia que está siendo actualmente muy explorada ya que permite utilizar ?a priori? el conocimiento disponible sobre absorción, metabolismo, biodisponibilidad y toxicidad de las moléculas a estudiar. El presente trabajo de tesis logró demostrar en modelos biofísicos y celulares que doxiciclina, una tetraciclina de segunda generación, es capaz de interferir en la agregación patológica de α-sinucleína formando especies de menor toxicidad e inhibiendo el proceso de nucleación-polimerización necesario para la propagación de la patología. La buena biodisponibilidad en cerebro de esta molécula, así como su potencial antioxidante y su baja toxicidad para tratamientos a largo plazo la convierten en un excelente modelo de reposicionamiento para la EP. Realizamos también un detallado estudio estructura-función de diferentes familias de antibióticos con capacidad de inhibir la agregación amiloide de proteínas y logramos descifrar un motivo estructural común a todas ellas que sentaría importantes bases para el diseño racional de fármacos. En base a este motivo estructural, analizamos diferentes análogos estructurales y pudimos encontrar que una tetraciclina químicamente modificada denominada CMT-3 posee mayor eficiencia antiagregante y neuroprotectora que doxiciclina, conservando la propiedad de atravesar con facilidad la barrera hematoencefálica. Esta molécula posee además la ventaja de interaccionar con especies fibrilares maduras de α-sinucleína, induciendo su desensamblaje. Esta propiedad, sumada a su baja actividad antibiótica le confieren propiedades únicas como antiagregante, antioxidante y antineuroinflamatoria que convierten a CMT-3 en un fármaco ideal para entrar a la fase de pruebas clínicas en EP. Los resultados presentados posicionan a la doxiciclina y CMT-3 como nuevas estrategias terapéutica en la EP y en otras sinucleinopatías. Fil: González Lizarraga, Maria Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; Argentina

[cat] El nostre grup ha identificat prèviament diferents gens i programes transcripcionals regulats per la proteïna p27. En aquest treball ens centrem en el paper de la proteïna p27 en la regulació a nivell transcripcional de l’expressió del gen que codifica per l’α-sinucleïna (gen SNCA), una proteïna molt important en el desenvolupament de la malaltia de Parkinson. Específicament, l’α-sinucleïna forma agregats en regions concretes del cervell. En aquest treball hem comprovat que les cèl·lules deficients de p27 presenten nivells més elevats d’α-sinucleïna. Per altra banda, en cervells de ratolins KO de p27 també observem un augment de l’expressió d’α-sinucleïna. Addicionalment, mitjançant la tècnica de silenciament gènic CRISPR/Cas9 vam obtenir línies cèl·lules KO de p27 i E2F4 i en aquestes cèl·lules també s’observa un augment de l’α-sinucleïna. Posteriorment, hem descrit, mitjançant experiments de ChIP i de luciferases, la regulació a nivell transcripcional de p27 del gen SNCA en associació a diferents factors de transcripció, C/EBPδ i E2F4/p130. En ambdós casos l’expressió de p27 reprimeix l’expressió del gen SNCA. A més a més, mitjançant shRNAs i la tècnica de Proximity Ligation Assay (PLA) hem observat com la disminució dels nivells de p27 i p130 també augmenten l’agregació de l’α-sinucleïna. També hem identificat CDK5 com una possible quinasa encarregada de fosforil·lar a p27 i exportar-ho al citoplasma on no pot exercir la seva funció com a regulador transcripcional. Finalment, mitjançant shRNAs vam disminuir els nivells de CDK5, en aquestes cèl·lules i amb la tècnica de PLA hem aconseguit veure com la supressió de CDK5 provoca un augment en l’agregació de l’α-sinucleïna. Com està descrit que CDK5 és capaç d’interaccionar amb els complexes E2F1/pRB, hem caracteritzat la unió de p27 a E2F1 mitjançant columnes cromatogràfiques i hem establert que E2F1 s’uneix a al extrem final de p27, entre els aminoàcids 160-198, de manera similar a la que s’uneix amb E2F4., [eng] Our group has identified different transcriptional programs regulated by the protein p27. In this work we focus on the role of p27 in the transcriptional regulation of the gene that encodes for α-synuclein (SNCA gene), a very important protein involved in Parkinson’s disease. Specifically, α-synuclein forms aggregates in specific regions of the brain. In this thesis, we found that p27-deficient cells have higher levels of α-synuclein. On the other hand, in brains of p27 KO mice we also observed an increase in α-synuclein levels in some brain regions. Additionally, using the CRISPR/Cas9 gene silencing technique we obtained KO cell lines of p27 and E2F4 and in these cell lines we also observe an increase of α-synuclein. Subsequently, through ChIP and luciferase assays, we described the transcriptional regulation of p27 of the SNCA gene in association with two different transcription factors, C/EBPδ and E2F4/p130. In both cases, p27 expression supresses SNCA expression. Furthermore, using shRNAs and the Proximity Ligation Assay (PLA) technique, we observed how the decrease of p27 and p130 protein levels also increased α-synuclein aggregation levels. We have also identified CDK5 as a kinase that phosphorylates p27 and cause it exportation to cytoplasm where it cannot function as a transcriptional regulator. Finally, using shRNAs we decreased the levels of CDK5 in these cells and with PLA we were able to see how the decrease of CDK5 levels causes an increase in α-synuclein aggregation levels. As it is described that CDK5 can interact with E2F1/pRb complexes, we have characterized the binding of p27 to E2F1 by affinity chromatography assays and we established that, as occurs with E2F4, E2F1 binds to the carboxyl moiety of p27, between aminoacids 160-170.

Objetivo. La presente investigación pretendió determinar la relación entre la expresión de la alfa sinucleina en sangre y el estreñimiento crónico en población residente en Bogotá, D.C. con consumo problemático de alcohol. Metodología. La población de estudio estuvo conformada por 62 individuos asignados a los grupos de acuerdo al puntaje obtenido mediante el cuestionario AUDIT (35 sujetos en el grupo de controles y 27 sujetos en el grupo de casos). Para el diagnóstico de estreñimiento se aplicaron los criterios de Roma IV y aspectos relacionados con la historia clínica. Los participantes tuvieron un periodo de abstinencia ≥ 48 horas, hasta la extracción de sangre periférica. La expresión del gen fue evaluada mediante qPCR y la cuantificación proteica por ELISA. Adicionalmente, se determinó la presencia de SNP’s, así como el estado de metilación en un segmento de la región promotora del gen SNCA, mediante secuenciación. Resultados. En este estudio se observó un incremento significativo en la expresión génica de ARNm de SNCA en individuos con problemas de consumo de alcohol, el cual no se relacionó con el diagnóstico positivo de estreñimiento crónico o por la presencia de los SNP’s rs261963 y rs2301134 en la región promotora del gen. Se evidenció un riesgo 4.8 veces mayor de presentar estreñimiento en personas con alto consumo de alcohol. Se encontraron 9 SNP´s en el segmento de la región promotora del gen SNCA, cuya frecuencia fue similar entre grupos de estudio y se identificó un SNP en la posición -2171, que no se encuentra reportado en GenBank para variantes clínicas y cuyo genotipo AT se relacionó con el aumento de ARNm de SNCA. Conclusión. En sujetos con problemas de consumo de alcohol, se evidenció sobreexpresión de ARNm de SNCA, lo cual no estuvo relacionado con el diagnóstico de estreñimiento crónico. Abstract: Objective. The present investigation aimed to determine the relationship between the expression of alpha synuclein in blood and chronic constipation in a population resident in Bogotá, D.C. with problematic alcohol consumption. Methodology. The study population consisted of 62 individuals assigned to the groups according to the score obtained through the AUDIT questionnaire (35 subjects in the control group and 27 subjects in the case group). For the diagnosis of constipation, the criteria of Rome IV and aspects related to the clinical history were applied. The participants had a period of abstinence ≥ 48 hours, until the extraction of peripheral blood. The expression of the gene was evaluated by qPCR and protein quantification by ELISA. Additionally, the presence of SNP's, as well as the methylation status in a segment of the promoter region of the SNCA gene, was determined by sequencing. Results. In this study, a significant increase in gene expression of SNCA mRNA was observed in individuals with alcohol consumption problems, which was not influenced by the positive diagnosis of chronic constipation or by the presence of rs261963 y rs2301134 SNPs in the promoter region of the gene. A 4.8 times higher risk of constipation was observed in people with high alcohol consumption. We found 9 SNPs in the segment of the promoter region of the SNCA gene, whose frequency was similar between study groups and a SNP was identified in position -2171, which is not reported in GenBank for clinical variants, and whose AT genotype was related to the increase of SNCA mRNA. Conclusion. In subjects with problems of alcohol consumption, overexpression of SNCA mRNA was evidenced, which was not related to the diagnosis of chronic constipation. Maestría

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais frequente. Com o aumento da esperança média de vida tem-se constatado o incremento da prevalência desta doença. Desde os anos 60 até aos dias de hoje, os tratamentos disponíveis têm como objetivo restabelecer os níveis de dopamina, aliviando somente a sintomatologia motora. Contudo, estes têm demonstrado eficácia limitada, dado que o seu uso a longo prazo está associado ao aparecimento de movimentos involuntários e espasmos. Surge, por isso, a necessidade de se desenvolverem estratégias terapêuticas mais eficazes. Durante muito tempo, as imunizações com vista a estimular respostas imunológicas foram aplicadas somente na prevenção de infeções microbianas. Investigações recentes sugerem que as imunoterapias destinadas a induzir ou suprimir o sistema imunológico podem ser usadas para combater doenças não infeciosas, como doenças neurodegenerativas, usando como alvo as proteínas agregadas enroladas incorretamente. A presença de inclusões intraneuronais de α-sinucleína é um dos hallmarks neuropatológicos da DP. Nos últimos anos, a comunidade científica tem investigado o papel da α-sinucleína e a sua relação com a fisiopatologia da DP, dada a hipótese de ser uma proteína priónica e estar associada com a neurodegenerescência progressiva característica desta doença. Nesse sentido, têm sido investigadas estratégias imunoterapêuticas, algumas das quais em fase de ensaios clínicos, que visam eliminar formas citotóxicas de α-sinucleína, interligando a função crucial da microglia no processo patológico. Nesta monografia abordam-se os estudos científicos que têm vindo a ser desenvolvidos no âmbito da aplicação da imunoterapia na doença neurodegenerativa de Parkinson, com vista a retardar a progressão da doença, assim como os resultados dos ensaios clínicos, alguns dos quais bastante promissores. Parkinson's disease is the second most frequent neurodegenerative disease. With the increase of the life expectancy, it has been observed the rise in the prevalence of Parkinson’s disease. Since the 1960s so far, available treatments aim to restore dopamine levels, relieving only motor symptomatology. However, these treatments have shown limited efficacy, since their long-term use is associated with the appearing of involuntary movements and spasms. There is, therefore, a need to find more effective treatments. For a long time, immunizations to stimulate immune responses were applied only in the prevention of microbial infections. Recent investigations suggest that immunotherapies designed to induce or suppress the immune system can be used to combat non-infectious diseases, as neurodegenerative diseases, by targeting aggregated proteins with incorrect folding. The presence of intraneuronal α-synuclein inclusions is one of the neuropathological hallmarks of Parkinson's disease. In recent years, the scientific community has investigated the role of α-synuclein and its relation to the pathophysiology of Parkinson’s disease, given the hypothesis of being a prion protein, associated with the progressive neurodegeneration characteristic of this disease. By this way, immunotherapeutic strategies, some of which in clinical trials, have been developed in order to eliminate cytotoxic forms of α-synuclein, linking the crucial function of microglia in the pathological process. This monograph aims to review the scientific studies that have been developed in the scope of the application of immunotherapy in neurodegenerative Parkinson's disease, in order to delay the progression of the disease, as well as the results of clinical trials, some of which have shown promise results.

Current understanding of the pathophysiology of Parkinson's disease suggests a key role of the accumulation of alpha-synuclein in the pathogenesis. This critical review highlights major landmarks, hypotheses and controversies about the origin and progression of synucleinopathy in Parkinson's disease, leading to an updated review of evidence suggesting the enteric nervous system might be the starting point for the whole process. Although accumulating and compelling evidence favors this theory, the remaining knowledge gaps are important points for future studies. RESUMO O atual entendimento sobre a fisiopatologia da doença de Parkinson (DP) sugere um papel central do acúmulo de alfa-sinucleína na patogenia da DP Esta revisão crítica revisita marcos, teorias e controvérsias a respeito da origem e progressão da sinucleinopatia, apresentando uma atualização das principais evidências sugerindo que o sistema nervoso entérico seria o local inicial deste processo. Apesar das evidências a favor desta teoria serem crescentes e instigantes, as lacunas de conhecimento a este respeito são importantes pontos para estudos futuros.

Tesis (Tecnólogo Médico) Los trastornos neurodegenerativos se definen como proteinopatías, donde se transforma una proteína funcional, en una con plegamiento anómalo, y la acumulación de ésta, conlleva a una pérdida neuronal progresiva. La enfermedad de Parkinson se caracteriza por la acumulación de la proteína sináptica α-sinucleína en neuronas dopaminérgicas; es el componente principal de los cuerpos de Lewy, marcador anatomopatológico de la enfermedad de Parkinson, su presencia se relaciona con la alteración de los procesos de transporte neuronal y axonal, agregados tóxicos, disfunción mitocondrial y muerte neuronal. α-sinucleína puede alterar su conformación y adquirir capacidad autoagregante, formando oligómeros que contienen hoja β. La importancia patogénica de esta proteína se basa en su capacidad de formar oligómeros que deterioran la transmisión sináptica y el funcionamiento neuronal, formando poros en la membrana, que aumentan la permeabilidad y alteran la homeostasis iónica celular, generando un efecto de degeneración y muerte neuronal. Es por esto, que esta unidad de investigación surge por la necesidad de establecer el efecto que ejercen los agregados proteicos sobre el citoesqueleto neuronal y el nivel de citotoxicidad que producen sobre las mismas células. Para esto, se utilizó una línea celular inmortalizada proveniente de neuroblastoma SH-SY5Y, sobre la cual se realizó marcaje inmunofluorescente con anticuerpos tubulina β-III, α-tubulina, y neurofilamentos. Esto no entregó evidencias observables morfológicamente sobre disrupción del citoesqueleto, sin embargo, estadísticamente se obtuvieron diferencias significativas, que indicaron cambios a nivel del citoesqueleto. En paralelo, se realizó la medición de la actividad de la enzima lactato deshidrogenasa (LDH) en el sobrenadante del cultivo celular, dicha medición concuerda con el análisis fluorescente del citoesqueleto, ya que se obtuvieron niveles elevados de LDH, indicando que los oligómeros inducen citotoxicidad.

Crescentes evidencias indicam que a susceptibilidade genética contribui para a etiologia da doença de Parkinson esporádica (DP). Variações genéticas no gene SNCA, responsável pela codificação da alfa-sinucleína, foram bem estabelecidas através de estudos de linkage e GWAS. Estudos em todo o mundo encontraram associação positiva com polimorfismos de nucleotídeo único (SNPs) no gene SNCA e o aumento do risco para DP. Além disto, variações no SNCA podem influenciar características ou fenótipos da DP esporádica. Este estudo teve como objetivo investigar associações entre os SNPs no gene SNCA e a sintomatologia motora e não-motora da DP em uma população brasileira. 206 sujeitos (grupo DP= 105 e grupo controle= 101) participaram do estudo. Os pacientes com DP foram recrutados no ambulatório de neurologia do Hospital Onofre Lopes, na cidade de Natal (RN). Foram utilizados questionários e escalas para avaliar o histórico de saúde, fatores ambientais, aspectos motores (Hoehn & Yarh e Unified Parkinson´s Disease Rating Scale), funcionais (Schwab & England), cognitivos (Bateria de Avaliação Frontal e Mini-Exame do Estado Mental) e emocionais (Inventário de Depressão de Beck e Inventario de Ansiedade de Beck) dos participantes. Também foi coletada uma amostra de sangue para a realização da extração do DNA e posterior genotipagem dos SNPs rs11931074, rs356219, rs2583988, rs2736990. Quanto a avaliação clínica, o grupo DP apresentou comprometimento motor de moderado a severo associado a uma moderada redução da capacidade funcional. Os déficits cognitivos foram mais presentes nos sujeitos com baixa escolarização. Os sintomas depressivos e ansiosos foram evidenciados com maior frequência e gravidade no grupo DP. Os dados da genotipagem mostraram todos os SNP mais frequentes no grupo DP, e a associação dos SNPs rs356219, rs2583988, rs2736990 aumentando o risco para DP foi confirmada. Também foi encontrada uma associação dos alelos de risco com a DP de início precoce. T-rs2583988, G-rs356219 e C-2736990 foram significativamente mais frequentes nos pacientes com alterações cognitivas quando comparados aos controles com a mesma condição. Além disto, a presença de alterações cognitivas foi mostrou-se um fator preditivo para a DP em um modelo de regressão logística. Este estudo é o primeiro a demonstrar a associação de polimorfismos no gene SNCA em uma população da América do Sul. Increasing evidence indicate that genetic susceptibility contributes to the etiology of sporadic Parkinson´s Disease (PD). Genetic variations in SNCA gene, which encodes alpha-synuclein protein, are already well established in linkage and GWAS studies. Worldwide studies have find positive association of single nucleotide polymorphism (SNP) in SNCA and the increase risk for PD. In addition, variants in SNCA can influence individual traits or phenotypes of sporadic PD. The present study investigated associations between SNPs in SNCA gene and motor and non-motor symptoms of PD in a Brazilian population. 206 subjects (PD group= 105 and Control group = 101) participated in this study. The patients with PD were recruited from the neurology clinic of Onofre Lopes University Hospital, in Natal (RN, Brazil). We used questionnaires and scales to evaluate the healthy history, environmental factors, motor (Hoehn & Yarh e Unified Parkinson´s Disease Rating Scale), functional (Schwab & England), cognitive (Frontal Assessment Battery and Mini Mental State Examination) and emotional (Beck Depression Inventory and Beck Anxiety Inventory) aspects of the subjects. We also collected a blood sample to DNA extraction and genotyping of SNPs rs11931074, rs356219, rs2583988 and rs2736990. Regarding clinical assessment, the PD group presented motor impairment associated to a moderate decrease of functional capacity. The cognitive impairments were more evident in individuals with low education. Depressive and anxiety symptoms had a higher frequency and severity in PD group. All SNPs were more frequent in PD patients (p

Altres ajuts: The authors thank Tom Yohannan for the editorial advice and the IBEC Nanotechnology Platform staff for their generous help. This research was supported by grants from CIBERNED (PI2014/02-4 (Rapid dementias), PRY-14-114 and PRY2016-02, La Caixa Obra Social Foundation and La Marató de TV3) to J.A.D.R. M.S.-F was supported by CIBERNED, A.H was supported by La Caixa Obra Social Foundation and L.U. was supported by a fellowship from the Marató TV3 foundation. The cellular prion protein, encoded by the gene Prnp, has been reported to be a receptor of β-amyloid. Their interaction is mandatory for neurotoxic effects of βamyloid oligomers. In this study, we aimed to explore whether the cellular prion protein participates in the spreading of α-synuclein. Results demonstrate that Prnp expression is not mandatory for α-synuclein spreading. However, although the pathological spreading of αsynuclein can take place in the absence of Prnp, αsynuclein expanded faster in PrPC-overexpressing mice.

Lewy body disorders (LBD) are common neurodegenerative diseases characterized by the presence of aggregated proportional to-synuclein in Lewy bodies and Lewy neurites in the central and peripheral nervous systems. The brains of patients with LBD often display other comor-bid pathologies, i. e. insoluble tau, beta-amyloid aggregates, TAR DNA-binding protein 43 (TDP-43) deposits, and argyrophilic grain disease (AGD). The incidence and physiological relevance of these concurrent pathological findings remain controversial. We per-formed a semiquantitative detailed mapping of proportional to-synuclein, tau, beta-amyloid (A beta), TDP-43, and AGD pathologies in 17 areas in 63 LBD cases (44 with Parkinson disease [PD], 28 with dementia, and 19 with dementia with Lewy bodies). APOE and MAPT genetic variants were also investigated. A majority of LBD cases had 2 or 3 concomitant findings, particularly Alzheimer disease-related pathology. Pathological stages of tau, beta-amyloid and proportional to-synuclein pathologies were increased in cases with dementia. A beta score was the best correlate of the time to dementia in PD. In addition, beta-amyloid deposition correlated with beta-synuclein load in all groups. MAPT H1 haplotype did not influence any assessed pathology in PD. These results highlight the common concurrence of pathologies in patients with LBD that may have an impact on the clinical expression of the diseases.

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

Parkinson’s Disease (PD) is a progressive neurodegenerative disorder which manifests as a motor disorder, characterized by slowness of movements, rigidity and tremor. Motor dysfunction of PD is due to dopamine loss in the neostriatum and due to the neurodegeneration of dopaminergic neuron’s cell bodies of the Substantia Nigra Pars Compacta (SNPC). Evidence suggests that chronic neuroinflammation, mediated by activated microglial and astroglial cells in the SNPC, could be essential for the degenerative process. It is well known that IGF1 plays a key role in neuroprotection in physiological conditions, both in health and disease. Previous studies of our group have shown that Rad-IGF ICV gene therapy had a restorative effect on motor skills of aged rats., Facultad de Ciencias Exactas

Tesis (Tecnólogo Médico mención Bioanálisis clínico, Inmunohematología y Banco de Sangre) La Enfermedad de Parkinson es un trastorno neurodegenerativo, caracterizado por la presencia de tres síntomas principales tales como: (i) rigidez muscular, (ii) bradicinesia y (iii) temblor en reposo, además, puede presentarse inestabilidad postural y disminución de la expresión facial en los pacientes. Epidemiológicamente, afecta al 2-3% de la población mayor a 65 años, elevándose a un 3-5% en mayores de 80 años, presentando predominancia en el sexo masculino, siendo infrecuente antes de los 50 años. Con respecto a la patogénesis de la enfermedad, se describe la presencia de inclusiones citoplasmáticas en neuronas dopaminérgicas ubicadas en la Sustancia nigra pars compacta, llamados cuerpos de Lewy, los cuales poseen como principal compuesto la α-sinucleína. Actualmente el tratamiento es la levodopa, la cual posee un enfoque paliativo de la enfermedad, debido a que disminuye la sintomatología clásica, sin embargo, no evita la progresión de la enfermedad. Es por esto, que surge la necesidad de buscar nuevas alternativas al tratamiento actual, y que actúen evitando la progresión de la enfermedad, específicamente interactuando en el proceso de agregación, debido a los efectos citotóxicos de los oligómeros. Por lo tanto, en este trabajo se evaluó el nivel de inhibición de la agregación que presentaban seis polifenoles derivados del extracto del Rosmarinus officialis, realizando ensayos de agregación in vitro utilizando α-sinucleína recombinante, siendo monitoreados por Tioflavina-T (ThT) luego de 24 horas de incubación. Se realizó un screening de las seis moléculas, realizando posteriormente un ensayo de dosis-respuesta de la molécula que presentó mayor inhibición, para luego separar las estructuras formadas en presencia y ausencia del polifenol mediante cromatografía por exclusión de tamaño. A su vez, se comparó el efecto de la molécula en presencia de un inductor de la agregación, obteniendo diferencias significativas entre ambas condiciones.

La enfermedad de Parkinson es una patología neurodegenerativa que afecta al 1% de la población mayor de 60 años. Se caracteriza por déficits motores, temblor en reposo y una pérdida selectiva de las neuronas dopaminérgicas de la Substantia Nigra Pars Compacta. Otro rasgo típico es la aparición de agregados proteicos conocidos como cuerpos de Lewy cuyo principal componente es una pequeña proteína, α-sinucleína. Esta proteína se une a las membranas lipídicas y en su conformación de α-hélice podría modular la fusión de las vesículas sinápticas con las membranas pre-sinápticas e influiría en la liberación de neurotransmisores. En condiciones patológicas, α-sinucleína sería capaz de unirse a las mitocondrias y se ha propuesto que esta unión provocaría disfunción mitocondrial y estrés oxidativo que colaborarían en el desarrollo de la enfermedad. La hipótesis de este trabajo propone que AS se incorpora a la mitocondria y se distribuye en sus distintos compartimientos. En esas localizaciones, esta proteína generaría disfunción mitocondrial incidiendo negativamente sobre distintos parámetros metabólicos, sobre la dinámica mitocondrial y la autofagia. En el presente trabajo se estudió la localización específica de α-sinucleína en mitocondrias aisladas de cerebro de rata. Se emplearon técnicas de microscopía de fluorescencia, microscopía electrónica de transmisión, Transferencia de energía de resonancia de Förster (FRET), fraccionamientos celulares y mitocondriales, western blots y distintos ensayos bioquímicos. Los resultados obtenidos en mitocondrias aisladas mostraron que AS 1μM se asocia a la mitocondria, mayoritariamente en la membrana externa y AS 10μM y 50 μM se encuentra principalmente internalizada. La microscopía electrónica confirmó la localización de α-sinucleína en el interior de la mitocondria y también en la periferia. Mediante un experimento de FRET se demostró un posible rol para el transportador de membrana externa TOM en la incorporación de AS. El análisis de la funcionalidad mitocondrial reveló que α-sinucleína altera el consumo de O2, elpotencial de membrana, la producción de ATP y aumenta la generación de especies reactivas de oxígeno. Por otra parte, se utilizaron células SH-SY5Y transfectadas con α-sinucleína para estudiar el efecto sobre la viabilidad celular, la producción de ATP, la generación de especies reactivas de oxígeno, la dinámica mitocondrial y la autofagia. Los resultados revelaron una disminución significativa de la viabilidad, en paralelo a una disminución en la producción de ATP y generación aumentada de especies reactivas. Además, α-sinucleína indujo disipación del potencial de membrana mitocondrial, desbalance de los procesos de fisión-fusión y desencadenamiento de la autofagia y mitofagia. Estos últimos procesos actuarían como un mecanismo de rescate celular disminuyendo la muerte y eliminando la mitocondrias dañadas. Este trabajo agrega evidencia novedosa al conocimiento de la localización de α-sinucleína en los distintos compartimientos mitocondriales, sus efectos sobre la funcionalidad mitocondrial y la relevancia de la disfunción de la organela en el destino celular. De este modo representan una contribución a la comprensión de los procesos que llevan a la patogénesis de la Enfermedad de Parkinson. Parkinson disease is a neurodegenerative pathology of the central nervous system affecting 1% of the population over 60 years old. This disease is characterized by motor deficiency, rest tremor, and the selective loss of dopaminergic neurons of the Substantia Nigra Pars Compacta. Another typical feature is the occurrence of protein aggregates known as Lewy’s bodies. The main component of Lewy’s bodies is a small protein named α-synuclein. This protein binds to lipidic membranes. The α-helix conformation could regulate the fusion between synaptic vesicles and pre-synaptic membranes modulating the neurotransmitter release. In pathological conditions, α-synuclein is associated to mitochondria. This interaction would trigger mitochondrial impairment and reactive oxygen species generation both events involved in the development of Parkinson's disease. This study hypothesizes that α-synuclein could be incorporated into the mitochondria and distribute in their different compartments. In these locations, this protein would be able to impair the mitochondrial function implying processes such as metabolic imbalance, mitochondrial dynamics and autophagy. The present work studies the specific location of α-synuclein in isolated rat brain- mitochondria. Techniques of fluorescence microscopy, transmission electron microscopy, Förster Resonance Energy Transfer (FRET), cellular and mitochondrial fractionation, western blots and several biochemical assays were used. Results showed that α-synuclein 1μM was associated to mitochondria mainly on the outer membrane while α-synuclein 10μM and 50μM were internalized. Electron microscopy supported α-synuclein location inside the mitochondrion and also in the periphery. A possible role for the transporter outer membrane (TOM) in the incorporation of α-synuclein to mitochondria was suggested by FRET assay. The analysis of mitochondrial functionality revealed that α-synuclein alters O2consumption, mitochondrial membrane potential and ATP production. In addition, an increase in the reactive oxygen species generation was observed. On the other hand, employing a cellular experimental model (SH-SY5Y cells transfected with α-synuclein) we demonstrated that this protein diminished cell viability and reduced ATP production with a concomitant increase in reactive oxygen species generation. In addition, α-synuclein was able to trigger mitochondrial membrane dissipation, imbalance in fission-fusion processes, autophagy and mitophagy. Both autophagy and mitophagy could act as rescue cellular mechanisms by decreasing cell death and damaged mitochondria. Taken together, these results add new evidence to the knowledge of mitochondrial specific α-synuclein location, the effects of this protein on the mitochondrial functionality and the relevance of this organelle dysfunction on cell viability. Thereby, these studies represent a contribution to understand the processes leading to development of Parkinson Disease. Fil: Martínez, Jimena Hebe. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

26 p., La enfermedad de Parkinson es la segunda enfermedad neurodegenerativa más frecuente tras el Alzheimer, y es el trastorno del movimiento más frecuente en la actualidad. La causa de esta enfermedad actualmente es desconocida, lo que dificulta la búsqueda de tratamientos efectivos. En este trabajo me he planteado como objetivo principal la revisión de los mecanismos fisiopatológicos implicados en la enfermedad, profundizando en algunas hipótesis que podrían ser la causa de su desarrollo. Se pueden distinguir 2 grandes mecanismos fisiopatológicos: la formación de agregados de alfa-sinucleína y la disfunción mitocondrial. La agregación de alfa-sinucleína da lugar a los llamados cuerpos de Lewy, característicos de la enfermedad de Parkinson. Este proceso se debe tanto a mutaciones genéticas, esencialmente de los genes A30P, E43K y A53T, como a factores ambientales como pesticidas, iones metálicos u otros elementos. Por otro lado se encuentra la disfunción mitocondrial, que provoca un daño celular que puede conducir a la apoptosis de las neuronas dopaminérgicas. Existen también diversos factores que pueden conducir a ella, tanto ambientales como genéticos. En la regulación mitocondrial tiene gran importancia las mutaciones de PINK1 y parkina, pudiendo verse alterada su función en la enfermedad de Parkinson., Parkinson’s disease is the second most common neurodegenerative process in our society, only behind Alzheimer’s disease, and the commonest motor disorder. The origin of Parkinson’s is unknown and so the treatment is not efficacious. In this essay, I have reviewed recent bibliography on the physio-pathological mechanisms involved in the generation and development of this disease, with a special focus on the two main hypothesis currently studied: (i) the mechanisms involved in the generation of alpha-synuclein aggregations and (ii) the mitochondrial dysfunction. Alpha-synuclein aggregations are associated to Lewy bodies which, in turn, are a type of pathological protein structure observed in brain lesions of Parkinson’s disease. The origin of this disorder is not well known, though different evidence includes genetic mutations, involving A30P, E43K and A53T genes, and environmental elements, such as pesticides, metals and other. On the other hand, mitochondrial dysfunction might cause cellular damage leading to the apoptosis of dopaminergic neurones. The source of this problem is currently focused, as well, in external factors, but also in mutations of PINK1 and parkin enzymes., Grado en Farmacia

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INTRODUCCIÓN: Las sinucleinopatías son enfermedades neurodegenerativas que se definen por la presencia de agregados anómalos de alfa sinucleína (AS) intraneuronales (principalmente cuerpos de Lewy-LB) o gliales. Clínicamente se caracterizan por la presencia de los llamados síntomas motores (parkinsonismo) junto con los no motores, entre los que se encuentran los síntomas de disautonomía. El sustrato de los síntomas de disfunción autonómica de la sinucleinopatía más frecuente, la enfermedad de Parkinson (PD), no está claro. Se piensa que la disautonomía cardiovascular es una de las primeras alteraciones que ocurren en la PD y que también el tracto gastrointestinal se encuentra alterado desde el inicio más temprano. Esta hipótesis está basada en hallazgos histopatológicos, estudios epidemiológicos y funcionales. La detección post-mortem de depósitos anómalos de AS en pacientes sin sintomatología motora ha permitido acuñar el término de “enfermedad de cuerpos de Lewy incidentales” (iLBD). El hallazgo post-mortem de cuerpos de Lewy no se considera un simple marcador de envejecimiento celular. En los últimos años se está trabajando en detectar en vida a sujetos con cuerpos de Lewy incidentales sin clínica sugestiva de enfermedad neurológica para poder confirmar o descartar su progresión a una sinucleinopatía clínica. OBJETIVOS: Describir las características del sistema nervioso autónomo en la enfermedad de Parkinson y otras sinucleinopatías, partiendo de la hipótesis de que en las enfermedades neurodegenerativas asociadas a cuerpos de Lewy a nivel del SNC existe una afectación generalizada del SNAP. Apoyar que la alteración en el SNAP permite la distinción entre enfermedad de Parkinson y otros parkinsonismos no asociados a cuerpos de Lewy. RESULTADOS: Realizamos un estudio post-mortem en el que todos los sujetos con confirmación patológica de tener una enfermedad ligada a LB incluyendo 1 caso con iLBD tenían agregados de AS en el sistema nervioso autónomo periférico (pANS) con un gradiente descendente cráneo-caudal de patología en la cadena simpática y en el tracto gastrointestinal. En un segundo estudio exponemos que los agregados de AS pueden ocurrir in vivo en el pANS epicárdico de sujetos sin parkinsonismo, sugiriendo el diagnóstico de iLBD. La presencia en algunos de estos sujetos de síntomas no-motores como sueños vivos y estreñimiento que aparecen también en la PD premotora apoya esta interpretación. Por último, un tercer estudio sugiere que el uso del 123I-MIBG SPECT cardiaco podría ayudar en el diagnóstico diferencial entre los pacientes PD y otros parkinsonismos en los que el diagnóstico quede incierto a pesar de otros estudios. CONCLUSIONES: En las enfermedades asociadas a agregados patológicos de alfa sinucleína en forma de cuerpos y neuritas de Lewy a nivel del sistema nervioso central existe depósito de estos agregados en el sistema nervioso autónomo periférico que no aparece en otras enfermedades neurodegenerativas. El plexo cardíaco, ganglio estrellado y cadena simpática ganglionar se encuentran afectados por depósitos de alfa sinucleína en todos los sujetos con confirmación neuropatológica de enfermedad de Parkinson, demencia con cuerpos de Lewy y cuerpos de Lewy incidentales. La distribución de agregados de alfa sinucleína en cadena simpática y tracto gastrointestinal presenta un gradiente descendente cráneo-caudal. Hasta un 8% de sujetos asintomáticos presentan en vida agregados de alfa sinucleína a nivel del plexo cardiaco. Ello sugeriría que estos sujetos podrían encontrarse en una etapa pre-motora de una sinucleinopatía. Dada la frecuente afectación del pANS cardiaco, el estudio funcional de la inervación autonómica cardiaca mediante SPECT miocárdico con 123I-MIBG podría ser de utilidad en el estudio del parkinsonismo vascular cuando se sospeche una enfermedad de Parkinson coexistente., INTRODUCTION: Alphasinucleinopathies are neurodegenerative disorders defined by the presence of abnormal alpha-synuclein (AS) aggregates. These aggregates can be localized intraneuronally ( mainly Lewy bodies, LB) or in the neuroglia. These disorders are clinically characterized by motor symptoms (parkinsonism) and other so called “non-motor symptoms”, including here dysautonomic features. The final cause of dysautonomic dysfunction in the most common synucleinopathy (Parkinson´s disease, PD) is still unclear. However, cardiovascular dysautonomia could be one of the first features of PD. Also, gastrointestinal tract is thought to be involved even in early PD. This idea is supported by histopathological studies, epidemiologic research and functional studies. AS aggregates can be also detected post-mortem in subjects without clinical parkinsonism, who are now known as subjects affected from “incidental Lewy body disease” (iLBD). Several study groups are trying to detect living iLBD subjects in order to study their progression, if any, to clinical synucleinopahies. OBJECTIVES: To describe the characteristic of the autonomic nervous system (ANS) in PD and other synucleinopathies based on the hypothesis of a widespread ANS involvement in diseases with presence of LB at central nervous system (CNS) level. To describe the utility of applying functional ANS studies as a tool in the differential diagnosis of synucleinopathies. RESULTS: We described in a post-mortem study that every subject with confirmed diagnosis of a LB-related disease (including one iLBD) presented abnormal AS aggregates at peripheral ANS level. On our second paper, we described in vivo AS epicardic fat involvement in subjects without parkinsonism, that is, living iLBD subjects. Some of these subjects also presented with non-motor symptoms suggestive of PD such as constipation and vivid dreams. Our third study supports the role of cardiac 123I-MIBG SPECT as an useful tool in the differential diagnosis between PD and vascular parkinsonism. CONCLUSIONS: If a disorder is related to abnormal CNS LB or Lewy neurites, the peripheral ANS is going to be also involved by abnormal AS aggregates. Cardiac plexus, stellate ganglia and sympathetic chain are involved by AS aggregates in every subject with the diagnosis of PD, dementia with Lewy bodies and iLBD. Up to 8% of asymptomatic subjects could present in vivo AS aggregates in cardiac plexus. Due to the high prevalence of cardiac ANS in synucleinopathies, its functional study using 123I-MIBG SPECT could be useful in the differential diagnosis of vascular parkinsonism if a co-existent PD is suspected.

Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES A Doença de Parkinson (DP) está associada a sintomas motores e à perda de neurônios dopaminérgicos na via nigroestriatal. A proteína alfa-sinucleína é o principal componente dos corpos de Lewy, marcadores biológicos da doença, e tem sido associada a casos de Parkinson hereditário. Recentemente, a estimulação da medula espinhal (EME) tem se mostrado um método de neuromodulação efetivo em aliviar os sintomas parkinsonianos em modelos animais e pacientes humanos da DP. Nesse projeto, caracterizamos os efeitos motores e eletrofisiológicos da expressão de alfa-sinucleína na substância nigra de ratos. Além disso, investigamos os efeitos da administração de AMPT, L-dopa e da aplicação de EME nesse modelo. Método: Ratos Sprague-Dawley receberam injeção unilateral de vetor viral vazio ou para expressar alfa-sinucleína na substância nigra e foram avaliados semanalmente na tarefa do campo aberto e do cilindro. Um grupo separado de animais implantados com matrizes bilaterais de eletrodos no córtex motor e no estriado foram registrados semanalmente no campo aberto, durante as sessões de EME e nos experimentos farmacológicos. Resultados: A expressão de alfa-sinucleína resultou em assimetria motora, observada na redução do uso da pata contralateral na tarefa do cilindro. Os animais apresentaram aumento da atividade de potencial de campo local estriatal em beta após três e quatro semanas de expressão de alfa-sinucleína, que desapareceu a partir da quinta semana. A administração de AMPT resultou em um quadro parkinsoniano severo, com redução da atividade locomotora e significativo aparecimento do pico de atividade oscilatória em beta no estriado e no cortex desse modelo. A EME mostrou-se efetiva em aliviar a assimetria motora em longo prazo, mas não reduziu as oscilações corticostriatais de baixa frequência observadas 24 hs após a administração de AMPT. Essas oscilações foram atenuadas pela administração de L-dopa que, de forma semelhante à EME, não foi efetiva em restaurar a atividade locomotora dos animais durante esse quadro de depleção severa de dopamina. Discussão: O modelo alfa-sinucleína da DP reproduz o prejuízo motor e a condição progressiva do processo neurodegenerativo. Nós demonstramos, pela primeira vez, que esse modelo apresenta também aumento da atividade corticostriatal oscilatória na banda beta compatível com as características da doença e que a EME tem efeito terapêutico sobre o sintoma motor desse modelo. Parkinson disease (PD) is associated with motor symptoms and dopaminergic cell loss in the nigrostriatal pathway. Alpha-synuclein is the major component of the Lewy bodies, the biological hallmarks of disease, and has been associated with familial cases of PD. Recently, the spinal cord stimulation (SCS) showed to be effective to alleviate the Parkinson symptoms in animal models and human patients. In this project, we characterized the motor and electrophysiological effects of alpha-synuclein overexpression in the substantia nigra of rats. We further investigated the effects of spinal electrical stimulation, AMPT and L-dopa administration in this model. Method: Sprague-Dawley rats were injected with empty viral vector or the vector carrying the gene for alpha-synuclein in the substantia nigra, and were tested weekly for 10 weeks in the open field and cylinder tests. A separated group of animals implanted with bilateral electrode arrays in the motor cortex and the striatum were recorded in the open field, during the SCS sessions and the pharmacological experiments. Results: Alpha-synuclein expression resulted in motor asymmetry, observed as the reduction in use of contralateral forepaw in the cylinder test. Animals showed an increase of local field potential activity in beta band three and four weeks after the virus injection, that was not evident after the 5th week. AMPT resulted in a sever parkinsonian state, with reduction in the locomotor activity and significant peak of oscillatory activity in cortex and striatum. SCS was effective to alleviate the motor asymmetry at long term, but did not reduce the corticostriatal low frequency oscillations observed 24 hs after the AMPT administration. These oscillations were attenuated by L-dopa that, even as SCS, was not effective to restore the locomotor activity during the severe dopaminergic depletion period. Discussion: The alpha-synuclein model reproduces the motor impairment and the progressive neurodegenerative process of PD. We demonstrated, by the first time, that this model also presents the increase in low frequency oscillatory activity in the corticostriatal circuit, compatible with parkinsonian condition; and that SCS has a therapeutic effect on motor symptom of this model.