Protein aggregation, oligomer and fibril formation is one of the dominant characteristics in the pathogenesis of a number of neurodegenerative diseases, such as Alzheimer’s disease (AD). Inhibition of toxic oligomer and fibril formation is one of the approaches to find potential drug candidates for AD. Additionally, early diagnosis of these amyloid species can provide mechanistic understanding of protein aggregation and thus can pave the way for preventing the onset of AD. The aim of this dissertation was 1) to explore the effects of charged cholesterol derivatives on the aggregation kinetic behavior of Amyloid-β40 (Aβ40), 2) to probe Aβ40 oligomer and amyloid formation in vitro using gold nanoparticles (AuNPs), and 3) to monitor the kinetic effect of various natural product molecules on Aβ40 aggregation in vitro. In the first chapter, a general introduction about AD as an amyloidogenic disease, amyloid cascade hypothesis, and the manipulation of Aβ peptides aggregation kinetics using different approaches was presented. In the second chapter, we studied the effects of oppositely charged cholesterol derivatives on the aggregation kinetics of Aβ. In the third chapter, we developed a gold nanoparticles (AuNPs) assay to probe Aβ40 oligomers and amyloid formation. In chapter IV, we monitored the effects of various small molecules on the aggregation kinetics of Aβ40. In chapter V, we discussed the methods and experimental details. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2018. / FAU Electronic Theses and Dissertations Collection

Les émulsions laitières sont des systèmes thermodynamiquement instables qui doivent résister aux contraintes technologiques (chauffage, congélation) appliquées lors de leur fabrication ou usage. Les émulsions riches en protéines de lactosérum sont particulièrement sensibles et l’emploi d’additifs alimentaires est un moyen de ralentir leur déstabilisation. Dans l’objectif d’offrir des produits 100 % lait aux consommateurs, concevoir des émulsions, riches en protéines de lactosérum, sans additifs alimentaires et stables aux traitements technologiques, constitue un réel challenge. La stratégie employée dans ce projet de thèse a été de combiner les propriétés des agrégats de protéines de lactosérum et des caséines pour stabiliser des émulsions aux cours des traitements technologiques sur une large gamme de concentration.Des émulsions ont été préparées avec des agrégats de protéines de lactosérum de structure différente et avec différents ratios agrégats/caséines. Quelle que soit leur structure, la présence d’agrégats à la surface des globules gras déstabilise l’émulsion (gélification /séparation de phase) alors que dans la phase dispersante ceux-ci sont stables aux traitements technologiques. A l’inverse, les émulsions dont la surface des globules gras est recouverte de caséines sont très stables aux traitements technologiques. Ainsi, il est possible de moduler la stabilité des émulsions riches en protéines de lactosérum aux cours des traitements technologiques en exploitant les propriétés des agrégats et des caséines et en contrôlant leur répartition entre la surface des glo / Dairy emulsions are thermodynamically unstable systems, which have to be resistant to the technological treatments (heating, freezing/thawing) applied during their manufacture or use. Whey protein-rich emulsions are particularly sensitive to technological treatments and instabilities are currently tackled by the use of non-dairy additives. With aim to offer products that are more natural to consumers (additive-free), the preparation of whey protein-rich emulsions without additive and stable during technological treatments constitutes a major challenge for dairy companies. The strategy adopted during this thesis was to combine the properties of the whey proteins aggregates and caseins in order to stabilize emulsion during technological treatments in a large range of protein concentrationsEmulsions were prepared with various whey protein aggregates and various whey protein aggregates/caseins ratio. Whatever the whey protein aggregates, their presence at the fat droplet surface destabilize the emulsions (gelation/phase separation) whereas they are stable in the continuous phase of the emulsions during technological treatments. In contrast, emulsions are extremely stable during technological treatments when caseins fully cover the fat droplet surface. The results obtained highlighted the possibility of modulating the stability during technological treatments of whey protein-rich emulsions by combining the properties of the whey protein aggregates and the caseins and by controlling their repartition between the fat droplet surface and the continuous phase of the emulsion.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23520号 / 農博第2467号 / 新制||農||1086(附属図書館) / 学位論文||R3||N5351(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 谷 史人, 教授 保川 清, 准教授 中川 究也 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

E3-Ubiquitin-Ligasen übertragen Ubiquitin auf die von ihnen gebundenen Substratproteine. Durch diese Ubiquitinierung werden Proteine für den kontrollierten Abbau im Ubiquitin-Proteasom-System markiert. Dieser Prozess beeinflusst aber auch die Aktivität verschiedener Signalwege, die Lokalisation von Proteinen oder die strukturelle Integrität zellulärer Komponenten. MuRF1, MuRF2 und MuRF3 sind E3-Ubiquitin-Ligasen, die hauptsächlich in quergestreifter Muskulatur exprimiert werden. Von MuRF1 ist bereits bekannt, dass es u. a. über die Ubiquitinierung von Myosinen und deren anschließender Degradation an der Entwicklung der Herz- und Skelettmuskelatrophie beteiligt ist. Da das Wissen über MuRF2 und MuRF3 in diesem Zusammenhang noch begrenzt ist, sollte die Auswirkung der kombinierten Keimbahndeletion von MuRF2 und MuRF3 in einem Mausmodell untersucht werden. Der Doppelknockout (DKO) von MuRF2 und MuRF3 führte zu Veränderungen der Morphologie und der Funktionsfähigkeit der Skelett- und Herzmuskulatur. In Skelettmuskelfasern kam es zur Ablagerung myosinhaltiger Proteinaggregate, zu einer Zunahme an langsam kontrahierenden Muskelfasern sowie zum Auftreten von Myozyten mit zentral gelegenen Nuclei als Anzeichen von Regenerationsprozessen. Isolierte Skelettmuskeln von DKO-Mäusen entwickelten eine geringere maximale spezifische Kraft als Muskeln aus Kontrolltieren. Ihre Herzen waren morphologisch unauffällig. Dennoch waren die Kontraktion des linken Ventrikels und das Schlagvolumen reduziert. Darüber hinaus zeigten isolierte Kardiomyozyten Beeinträchtigungen der Kontraktionsfähigkeit und der Kalziumströme in vitro. Eine massenspektrometrische Untersuchung ergab, dass in den Muskeln der MuRF2/3-DKO-Mäuse im Vergleich zu den Kontrollmäusen 12 Proteine in erhöhter Menge vorhanden waren. Eine Anreicherung von MAPKAP-K3, einem dieser Proteine, und von MAPKAP-K2 konnte im Western Blot von Proteinlysaten aus Skelettmuskeln und dem Herz der MuRF2/3-DKO-Mäuse detektiert werden. / E3 ubiquitin ligases attach the small modifier ubiquitin to their substrate proteins. This ubiquitin-tag not only marks proteins for the proteasome dependent degradation, but also influences the activity of signalling pathways, the localisation of proteins or the structural integrity of cellular components. MuRF1, MuRF2, and MuRF3 are E3 ubiquitin ligases predominantly expressed in striated muscles. MuRF1 is involved in cardiac and skeletal muscle atrophy by mediating proteasome-dependent degradation of myosins. The knowledge about MuRF2 and MuRF3 in this context is limited. Therefore, a mouse model was used to analyse the impact of the combined deletion of MuRF2 and MuRF3. The double knockout (DKO) of MuRF2 and MuRF3 influenced the structure and function of skeletal and cardiac muscle. Skeletal muscle fibres exhibited myosin-containing protein aggregates, a fibre-type shift towards slow fibres, and myoycytes with central nuclei which is an indication of regeneration. Maximal force development was reduced in isolated hindlimb muscles M. soleus and M. extensor digitorum longus of MuRF2/3-DKO mice. Hearts were morphologically normal. No protein aggregates or signs of fibrosis were detected. However, heart performance was impaired. The contractibility of the left ventricle and the ejection fraction were reduced. Isolated cardiomyocytes showed a diminished contractibility. Furthermore, their speed of contraction and relaxation was reduced and they had impaired calcium transients. Mass spectrometric analysis of muscle lysates identified 12 enriched proteins in MuRF2/3-DKO muscles. Western Blot analysis confirmed that MAPKAP-K3, one of these proteins, and MAPKAP-K2 were enriched in lysates of skeletal muscles and left ventricles of MuRF2/3-DKO mice. Further investigations will show how MAPKAP-K2- and MAPKAP-K3-signalling pathways are involved in the development of the MuRF2/3-DKO-phenotype.

Les Tunneling nanotubes (TNT) sont des protrusions cellulaires à base d'actine qui médient la communication cellulaire en transférant des cargos cellulaires. Les différents types de communication intercellulaires sont de plus en plus considérés comme des cibles potentielles pour le traitement de différentes maladies, telles que les maladies infectieuses liées aux virus et bactéries, les cancers ou les maladies neurodégénératives. Des études récentes ont mis en évidence un mécanisme de propagation d'agrégats protéiques ressemblant à la propagation du prion dans diverses maladies neurodégénératives non infectieuses telles que la maladie d'Alzheimer (AD), la démence frontotemporelle (FTD), la maladie de Parkinson (PD) et la maladie de Huntington. Ces maladies se caractérisent par l'accumulation de protéines mal repliées dans le cerveau des patients. Ainsi, on peut envisager de nouvelles stratégies thérapeutiques pour bloquer la propagation des protéines anormales dans tout le cerveau. Il a été démontré que les TNT pourraient jouer un rôle essentiel dans la propagation des agrégats de prions au sein du système nerveux central (SNC) et périphérique. Par conséquent, l'étude du mécanisme de la formation de TNT pourrait fournir de nouvelles idées sur le mécanisme de propagation de la maladie et de nouvelles cibles thérapeutiques. L'objectif de ma thèse était d'étudier le rôle du transfert des agrégats de protéines par les TNT entre les cellules et d'étudier le mécanisme de formation des TNT. Dans notre laboratoire, nous avons déjà montré que les TNT permettent le transfert de prions entre les cellules. Dans la première partie de mon doctorat, j'ai confirmé que les transferts d'agrégats de prions entre les cellules de CAD neuronales se faisaient par les TNT à l'intérieur de vésicules endocytiques (Zhu et al., 2015). De plus, en collaboration avec un collègue, nous avons fourni des preuves que les agrégats de prions pourraient être transférés entre des astrocytes primaires et des neurones et que ce transfert était médié par un contact cellulaire (Victoria et al., 2016). J'ai également collaboré à une autre étude où nous avons montré que les agrégats d'α-synucléine (caractéristiques de la maladie de Parkinson) peuvent être transférés entre les cellules à l'intérieur des lysosomes, et que ce transfert intercellulaire est médié par les TNT (Abounit et al., 2016). Dans mon deuxième projet, afin d'étudier le mécanisme de la formation de TNT, j'ai effectué un crible à haut débit pour les Rab GTPase. J'ai trouvé que Rab8 et Rab11 peuvent favoriser la formation des TNT, et que les cascades Rab8-VAMP3, Rab11-ERM et Rab8-Rab11 sont impliquées dans la formation des TNT. Mes données suggèrent que la polymérisation de l'actine et le trafic de membranes sont impliqués dans la formation des TNT. Ces résultats permettent d'éclairer le mécanisme de la formation des TNT et de fournir des preuves moléculaires que les Rab GTPases régulent ce processus. / Tunneling nanotubes are actin-based cell protrusions that mediate cell-to-cell communication by transferring cellular cargos. The different types of intercellular communication are increasing by being considered as potential targets for the treatment of various diseases, such as infectious diseases linked to viruses and bacteria, cancers or neurodegenerative diseases. Recent studies have highlighted a prion-like mechanism of propagation of protein misfolding in a variety of common, non-infectious, neurodegenerative diseases such as Alzheimer’s disease (AD), Frontotemporal dementia (FTD), Parkinson’s disease (PD), and Polyglutamine (PolyQ) diseases, which are characterized by the accumulation of misfolded proteins in the brain of patients. Thus, new therapeutic strategies to block propagation of protein misfolding throughout the brain can be envisaged. It has been shown that TNTs might play a critical role in spreading of prion aggregates within the CNS and from the periphery. Therefore, the study of mechanism of TNT formation could provide new insights on the mechanism of disease propagation and novel therapeutic targets. The aim of my thesis was to study the role of TNT-mediate protein aggregates transfer between cells and to investigate the mechanism of TNT formation. In our lab, we already reported TNT mediate prion transfer between cells. In the first part of my PhD, I further confirmed that prion aggregates transfer between neuronal CAD cells through TNT inside endocytic vesicles (Zhu et al., 2015). Furthermore in collaboration with a colleague, we provided evidences that prion aggregates could transfer between primary astrocytes and neurons and the transfer was mediated by cell-to-cell contact (Victoria et al., 2016). I also collaborated to another study where we showed that α-synuclein aggregates (Parkinson’s disease) can transfer between cells inside lysosomes, and the intercellular transfer is mediated by TNTs (Abounit et al., 2016).In my second project, in order to investigate the mechanism of TNT formation, I performed a High-content screening of Rab GTPase. I found that Rab8 and Rab11 can promote TNT formation, that Rab8-VAMP3, Rab11-ERM and Rab8-Rab11 cascades are involved in TNT formation. My data suggests that both actin polymerization and membrane trafficking are involved in TNT formation. These results help to shed light on the mechanism of TNT formation, and provide molecular evidences that Rab GTPases regulate this process.

Under det här kandidatexamensarbetet har protein-nanofibers påverkan på material undersökts genom att jämföra fibrillerade filmer med ofibrillerade. Sojaproteinisolat fibrillerades under förhållandena pH 2 och 85 ◦C under minst ett dygn och de syntetiserade nanofibrerna analyserades med Thioflavin T (ThT) fluorescens och atomkraftsmikroskopi (AFM). Spektra från analysmetoden ThT fluorescens indikerade på förekomsten av β-flak och analyserna med AFM visade på att fibrerna hade en morfologi som är karakteristisk för protein-nanofibrer. Resultaten antyder att de parametrar som påverkar morfologin hos fibrerna är fibrilleringstid och typ av protein. De gjutna filmerna från fibrillära respektive ofibrillära proteiner var sammanhängande bortsett från vissa sprickor. Värdena på E-modulen från AFM visade att det fibrillerade materialet var mer heterogent än det ofibrillerade. Filmer med sammanhängande yta erhölls vid tillsats av det mjukgörande additivet glycerol. Slutligen, material av både fibrillär och ofibrillär form kan framställas, däremot krävs vidare forskning för att optimera materialens egenskaper. / During this bachelor thesis project, the impact of protein nanofibers on materials has been analysed by comparing films made from fibrillar and non-fibrillar protein. Fibrillation of soy protein isolate was performed during at least 24 hours at pH 2 and a temperature of 85 ◦C. Analysis of the nanofibers was made with Thioflavin T (ThT) fluorescence and atomic force microscopy (AFM). The spectra from ThT Fluorescens indicated the presence of β-sheets and AFM confirmed that the fibrils had a morphology that is characteristic of protein nanofibers. The results indicated that heating time and protein type were the parameters which had the largest impact on the morphology of the fibrils. The synthesised films from both fibrillar and non-fibrillar protein were coherent with exception of some cracks. The elastic modulus from AFM indicated that the fibrillar film was more heterogeneous compared to the non-fibrillar film. To attain coherent films, the plasticising agent glycerol was added. To summarise, both fibrillar as well as non-fibrillar materials were successfully synthesised, however, further research is necessary to optimise the properties of the material.

Nous avons cherché à évaluer l'impact de conditions opératoires pour la préparation d'agrégats protéiques et d'émulsions stabilisées par des protéines en vue de la protection de l'α-tocopherol, servant de modèle de molécules d'intérêt, hydrophobes et sensibles. Les matrices protéiques ont été formées à partir d'un concentrat de protéines de lactosérum (6 wt% de WPC, pH 6.5 et 65 à 75°C), en présence ou absence de 4% α-tocopherol. Le mélange (65°C -15 min) des protéines en solution sans ou avec α-tocopherol a donné lieu à la formation de particules avec modification de charge (de -42 à -51 mV) et de taille (de 183 à 397 nm). Ces paramètres ont diminué davantage sous l'effet d'homogénéisation sous haute pression à 1200 bar que à 300 bar, alors qu'une meilleure protection de l'α-tocopherol a été observée après 8 semaines conservation. Les mécanismes impliqués dans la formation des matrices protéiques correspondantes ont été décrits sur la base de procédés de dénaturation-agrégation de protéines sériques, à partir de résultats obtenus par calorimétrie différentielle à balayage (DSC), spectrofluorescence, diffusion multiple de la lumière et électrophorèse SDS-PAGE. Les matrices lipidiques ont été préparées à partir de phases aqueuses contenant (6 wt% or 3 wt% de WPC) et lipidiques (20 %) en présence ou absence de lécithines (1.5%) avec ou sans α-tocopherol (4%), et par application d'une première étape de dispersion (65°C - 15 min) suivie d'une homogénéisation sous pression à 300 ou 1200 bar. Les nanoparticles lipidiques formées à plus haute pression étaient de taille et concentration protéique de surface plus faibles et de degré d'encapsulation de l'α-tocopherol plus faible (près de 15 %). L'analyse par DSC en modes balayage et isothermes des particules lipidiques a montré que plus leur taille est faible, plus le sur-refroidissement est important, l'apparition des cristaux de matière grasse plus retardée, et leur développement à 4°C moins important. Ces effets sont accentués dans les gouttelettes contenant l'α-tocopherol. La diffraction aux grands et petits angles de rayons X (synchrotron Soleil), couplée à la DSC, a montré la co-existence des polymorphes 2Lα, 2Lβ' et 2Lβ dans toutes les émulsions, mais à des proportions différentes. Les cristaux 2Lβ étaient plus développés dans les gouttelettes de plus petite taille et contenant du tocopherol en présence de lécithins, celles qui présentaient la plus forte dégradation chimique d'α-tocopherol pendant une conservation à long-terme. / We investigated effects of processing conditions for the preparation of protein aggregates and protein-stabilized lipid droplets, as matrix carriers of sensitive lipophilic bioactive compounds, with α-tocopherol as a model. Protein-based matrices were formed from whey protein concentrate (6 wt% WPC, pH 6.5 and 65 to 75°C), in presence or absence of 4% α-tocopherol. Mixing the protein solutions without or with α-tocopherol (65°C for 15 min) led to changes in particle surface charges (from -42 to -51 mV) and sizes (from 183 to 397 nm). These parameters decreased more under further high pressure homogenisation at 1200 bar than 300 bar, in parallel with increased vitamin protection over 8 week's storage. Molecular mechanisms involved in formation of corresponding α-tocopherol-loaded protein matrix were described on the basis of heat- and high-pressure-induced whey protein denaturation and aggregation, as evidenced by differential scanning calorimetry (DSC), spectrofluorescence, multi-light scattering and SDS-PAGE electrophoretic patterns. Lipid-based matrices were developed from aqueous phases (80 wt%) containing WPC (6 wt% or 3 wt%) and lipid phases (20 wt%) in presence or absence of lecithins and/or 4% α-tocopherol, and by using a first dispersion step (65°C for 15 min) followed with HPH at 300 or 1200 bar. Our results showed that increasing HPH was accompanied by formation of lipid nanoparticles with decreasing size and protein surface concentration with an increase in α-tocopherol degradation (up to 15 wt% for 1200 bar). DSC in scanning and isothermal modes showed that reduction in lipid droplet size was accompanied by retardation in crystalline fat development under storage at 4°C, with further reduction in crystalline fat development along with further increase in supercooling for lipid droplets containing α-tocopherol. Fat polymorphism observed using time-resolved synchrotron X-ray scattering at wide and small angles (WAXS and SAXS) coupled with DSC, showed co-existence of 2Lα, 2Lβ' and 2Lβ polymorphs in all the emulsions, but at different proportions. It was observed that 2Lβ polymorphs were more prominent in lipid droplets with lower size and containing α-tocopherol in presence of lecithins that were shown to present the lowest long-term stability of α-tocopherol against chemical degradation.

Le mépliement et l'agrégation des protéines sont à l'origine de nombreuses maladies neurodégénératives, dont la maladie de Huntington (HD) et la maladie de Parkinson (PD). Même si l’agrégation de différentes protéines liées à des maladies est bien documentée, on en sait peu sur l'interaction entre les protéines mal repliées et les cellules neuronales, qui leur permettent de se propager et affecter différentes régions du cerveau. L'objectif de ma thèse était de générer des modèles cellulaires rapporteurs de la huntingtine et l’α-synucléine, protéines dont le mauvais repliement et l'agrégation sont à l'origine de HD et PD respectivement, et utiliser ces modèles cellulaires pour étudier les interactions entre les agrégats et des lignées cellulaires de mammifères. Notre but c’était de documenter les propriétés de liaison et d’absorption de ces agrégats par les cellules rapporteuses, et les conséquences de leur internalisation pour les cellules. Deux modèles cellulaires de neuroblastome (SH-SY5Y et Neuro2A) et un modèle de cellules d’ostéoblastome (U2OS) exprimant la protéine fluorescente ChFP ont été générés pour HD. Pour simuler ce qui se passe au sein de neurones réels, des cellules de neuroblastome ont été induites à se différencier. Des différences de fixation, internalisation, nucléation de la protéine endogène et localisation finale des agrégats de polyglutamine internalisés ont été observées entre les cellules différenciées et non différenciées. Des cellules rapporteuses U2OS ont été utilisées pour déterminer les différences d’infectiosité entre des fibres de HttExon1 assemblés en présence ou en l’absence de la protéine de choc thermique constitutivement exprimée chez l'Homme Hsc70. Hsc70 a un effet protecteur car il rend les fibres moins infectieuses pour les cellules de mammifères en culture. Enfin, un modèle cellulaire de neuroblastome (Neuro2A) rapporteur pour PD exprimant l’α-synucléine fusionnée à la protéine ChFP a été utilisé pour déterminer des différences de liaison, pénétration, absorption, nucléation de la protéine endogène et persistance entre deux polymorphismes d’α-synucléine générés par notre équipe. L'hétérogénéité observée dans différents patients souffrant de synucléinopaties pourrait s'expliquer par différents polymorphes d’assemblages protéiques d’α-synucléine présents dans les cerveaux des malades, ce qui doit être pris en compte pour les développements thérapeutiques futurs.Ces modèles cellulaires rapporteurs pour différentes maladies sont un système valable pour l'étude de différents processus cellulaires liés à l'interaction entre les protéines agrégées exogènes et des cellules de mammifères en culture. Nos résultats indiquent un mécanisme commun par lequel les différentes protéines agrégées peuvent interagir avec des cellules en culture: les protéines mal repliées exogènes sont capables de se lier à des membranes cellulaires, les pénétrer, entrer dans l'espace intracellulaire et recruter des protéines endogènes solubles. Même si cela semble être un mécanisme générique pour des protéines infectieuses telles que la α-synucléine ou la huntingtine, des lignées cellulaires avec différents phénotypes montrent différences de vulnérabilité à la présence de protéines agrégées. Ceci suggère la présence de récepteurs spécifiques à la surface de la cellule capables de reconnaître des structures de type amyloïde. D'autres études sont nécessaires pour déterminer la nature de ces récepteurs et si sa modulation pourrait être utile pour contrôler la propagation des ces maladies dans le cerveau. / Protein misfolding and aggregation are at the origin of many neurodegenerative diseases, including Huntington’s disease (HD) and Parkinson’s disease (PD). Even if the aggregation of different disease-related proteins is well documented, little is known about the interaction between those misfolded proteins and neuronal cells that allow them to spread and affect several regions of the brain. The objective of my thesis was to generate reporter cellular models of huntingtin and α-synuclein, proteins whose misfolding and aggregation are at the origin of HD and PD respectively, and use these cell models for studying the interactions between misfolded protein aggregates and mammalian cell lines. We aimed to document the binding and uptake properties of those aggregates by reporter cells and the consequences of their internalization for the cells. Two neuroblastoma cell models (SH-SY5Y and Neuro2A) and an osteoblastoma cell model (U2OS) expressing the fluorescent protein ChFP were generated as mammalian reporter cell lines for HD. To mimic what happens in real neurons, neuroblastoma reporter cells were induced to differentiate. Differences in binding, internalization, nucleation of the endogenous protein and final localization of the internalized polyglutamine aggregates were observed between differentiated and undifferentiated cells. U2OS reporter cells were used for determining differences in the infectivity of HttExon1 fibrils assembled in the presence or in the absence of the constitutively expressed heat shock protein Hsc70, suggesting a protective effect of Hsc70, since it renders the fibrils less infectious to mammalian cells. Finally, a neuroblastoma reporter cell model (Neuro2A) of PD expressing α-synuclein fused to the fluorescent and reporter protein ChFP was used to determine the different binding, penetration, uptake, nucleation of the endogenous protein and persistence properties of two α-synuclein polymorphs generated by our team. The heterogeneity observed in different patients suffering from synucleinopathies could be explained due to different α-synuclein assemblies present in diseased brains, what needs to be taken into account for future therapeutic developments. These reporter cellular models for different diseases are a valid system for the study of different cellular processes related with the interaction between exogenous aggregated proteins and mammalian cells in culture. Our results indicate a common mechanism by which different aggregated proteins can interact with cells in culture: exogenous misfolded proteins are able to bind cell membranes, penetrate them, enter the intracellular space and recruit endogenous soluble proteins. Even if this seems to be a generic mechanism for infectious proteins such as α-synuclein or huntingtin, different cell lines or cell phenotypes show distinct vulnerability to the presence of aggregated proteins. This strongly suggests the presence of specific receptors at the surface of the cell able to recognize amyloid-like structures. Further investigations are needed to determine the nature of these receptors and whether their modulation might be helpful for controlling the spread of these diseases within the brain.

A miosite por corpos de inclusão (inclusion body myositis - IBM), na sua forma esporádica, é considerada a miopatia adquirida mais comum após os 50 anos de idade. Embora seja incluída no grupo das miopatias inflamatórias, estudos recentes mostram um processo particular de degeneração muscular caracterizado por deposição anormal de agregados de proteínas nas fibras musculares e funcionamento anormal dos principais sistemas de degradação proteica. O objetivo deste estudo foi o de avaliar os aspectos clínicos, histológicos e imunoistoquímicos de pacientes com IBM. Avaliamos 18 casos com diagnóstico de IBM de dois dos principais centros de doenças neuromusculares do Brasil (25 biópsias musculares). Na tentativa de diferenciar os casos de IBM das outras miopatias inflamatórias, determinamos o padrão de expressão tecidual da p-tau (p62), alfa-sinucleína e TDP-43. Também foi avaliada a função lisossomal através da reação da fosfatase ácida (marcação da atividade lisossomal global) e determinação da marcação para LC3B (marcador de autofagia). Foi observado que a IBM predominou no sexo masculino (61% dos casos), da cor branca, com início das manifestações clínicas ao redor dos 59 anos de idade e os sintomas mais frequentes foram fraqueza muscular, instabilidade postural com quedas da própria altura, disfagia e perda ponderal, podendo ainda apresentar dispneia. O diagnóstico demorou em média 7,4 anos após o início dos sintomas e frequentemente esteve associada às seguintes comorbidades: hipertensão arterial sistêmica, diabetes mellitus tipo 2, osteopenia / osteoporose, dislipidemia e hiperuricemia / gota. O padrão de comprometimento muscular na IBM foi caracterizado por tetraparesia de predomínio proximal em membros inferiores e distal em membros superiores. Os valores séricos da creatinofosfoquinase em pelo menos uma das medições foram elevados em todos os pacientes, porém sem ultrapassar 10 vezes o limite superior da normalidade. O uso de imunossupressão não se mostrou eficaz nos pacientes com IBM. Os achados histológicos na IBM incluíram alterações distróficas variáveis com a presença de inflamação endomisial, assim como a ocorrência de vacúolos marginados, além da elevada frequência de alterações mitocondriais. Outros achados histológicos musculares característicos na IBM foram o aumento da atividade lisossomal (aumento global da marcação para fosfatase ácida), a presença de marcação positiva para beta-amilóide (marcação intra-vacuolar pelo vermelho-Congo), o aumento na degradação muscular (relacionada com ativação de LC3B, p-tau, e p62/SQSTM1) e a degeneração muscular (marcação para anti-phospo TDP-43 e para ?-sinucleína). Tais alterações apresentaram alta sensibilidade e especificidade. Sugerimos que a redução do critério de idade do início dos sintomas de mais de 45 anos para mais de 35 anos aumentaria a sensibilidade diagnóstica para os casos com IBM deste estudo de 83% para 100%. Com este estudo, foi possível caracterizar clínica e histológicamente pacientes com IBM em nosso meio, e fornecer indícios do benefício do uso de marcadores de degeneração e autofagia para o diagnóstico e para a determinação de vias ou sistemas celulares envolvidos na patogênese da doença / Sporadic inclusion body myositis (sIBM) is considered the most common acquired myopathy affecting adults aged over 50 years. Although included in the group of inflammatory myopathies, recent studies show a particular process of muscle degeneration characterized by abnormal deposit of protein aggregates in muscle fibers and abnormal operation of the main protein degradation systems. The aim of this study was to evaluate the clinical, histological and immunohistochemical patients with IBM. We evaluated 18 cases with IBM diagnostic of two of the main centers of neuromuscular diseases in Brazil (25 muscle biopsies). In an attempt to differentiate the IBM cases of other inflammatory myopathies, we determined the pattern of tissue expression of p-tau (p62), alfa-synuclein and TDP-43. Also evaluated the lysosomal function by acid phosphatase reaction (marking global lysosomal activity) and determining the markup for LC3B (autophagy marker). It was observed that IBM was predominant in males (61% of cases), white colored, with onset of clinical manifestations around 59 years old and the most common symptoms are muscle weakness, postural instability with high falls, dysphagia and weight loss, and may also present dyspnea. The diagnosis took an average of 7.4 years after the onset of symptoms and was often associated with the following comorbidities: hypertension, type 2 diabetes mellitus, osteopenia / osteoporosis, dyslipidemia and hyperuricemia / gout. The muscular damage pattern at IBM was characterized by tetraparesis predominantly proximal lower limbs and distal upper limbs. Serum creatine kinase levels in at least one of the measurements were elevated in all patients, but not exceeding 10 times normal. Immunosuppression was not effective in patients with IBM. The IBM histological findings included diversify dystrophic changes, endomysial inflammation, as well as the occurrence of rimmed vacuoles, in addition to high frequency of mitochondrial changes. Other characteristic muscle histological findings in IBM were increased lysosomal activity (overall increase in labeling for acid phosphatase), the presence of positive staining for beta-amyloid (intra-vacuolar by Congo red marking), increased muscle degradation (related to activation of LC3B, p-tau and p62 / SQSTM1) and muscle degeneration (marking for anti-phospo TDP-43 and ?-synuclein). Such changes have a high sensitivity and specificity. which makes these important complementary analyzes for accurate pathological diagnosis. We suggest that lowering the age of the onset of symptoms of greater than 45 years to older than 35 years would increase the diagnostic sensitivity for cases with IBM this study from 83% to 100%. With this study, it was possible to characterize clinically and histologically the patients with IBM in our centers, and provide evidence of the benefit of using degeneration and autophagy markers for diagnosis and for determining pathways or cellular systems involved in the pathogenesis of the disease

A miosite por corpos de inclusão (inclusion body myositis - IBM), na sua forma esporádica, é considerada a miopatia adquirida mais comum após os 50 anos de idade. Embora seja incluída no grupo das miopatias inflamatórias, estudos recentes mostram um processo particular de degeneração muscular caracterizado por deposição anormal de agregados de proteínas nas fibras musculares e funcionamento anormal dos principais sistemas de degradação proteica. O objetivo deste estudo foi o de avaliar os aspectos clínicos, histológicos e imunoistoquímicos de pacientes com IBM. Avaliamos 18 casos com diagnóstico de IBM de dois dos principais centros de doenças neuromusculares do Brasil (25 biópsias musculares). Na tentativa de diferenciar os casos de IBM das outras miopatias inflamatórias, determinamos o padrão de expressão tecidual da p-tau (p62), alfa-sinucleína e TDP-43. Também foi avaliada a função lisossomal através da reação da fosfatase ácida (marcação da atividade lisossomal global) e determinação da marcação para LC3B (marcador de autofagia). Foi observado que a IBM predominou no sexo masculino (61% dos casos), da cor branca, com início das manifestações clínicas ao redor dos 59 anos de idade e os sintomas mais frequentes foram fraqueza muscular, instabilidade postural com quedas da própria altura, disfagia e perda ponderal, podendo ainda apresentar dispneia. O diagnóstico demorou em média 7,4 anos após o início dos sintomas e frequentemente esteve associada às seguintes comorbidades: hipertensão arterial sistêmica, diabetes mellitus tipo 2, osteopenia / osteoporose, dislipidemia e hiperuricemia / gota. O padrão de comprometimento muscular na IBM foi caracterizado por tetraparesia de predomínio proximal em membros inferiores e distal em membros superiores. Os valores séricos da creatinofosfoquinase em pelo menos uma das medições foram elevados em todos os pacientes, porém sem ultrapassar 10 vezes o limite superior da normalidade. O uso de imunossupressão não se mostrou eficaz nos pacientes com IBM. Os achados histológicos na IBM incluíram alterações distróficas variáveis com a presença de inflamação endomisial, assim como a ocorrência de vacúolos marginados, além da elevada frequência de alterações mitocondriais. Outros achados histológicos musculares característicos na IBM foram o aumento da atividade lisossomal (aumento global da marcação para fosfatase ácida), a presença de marcação positiva para beta-amilóide (marcação intra-vacuolar pelo vermelho-Congo), o aumento na degradação muscular (relacionada com ativação de LC3B, p-tau, e p62/SQSTM1) e a degeneração muscular (marcação para anti-phospo TDP-43 e para ?-sinucleína). Tais alterações apresentaram alta sensibilidade e especificidade. Sugerimos que a redução do critério de idade do início dos sintomas de mais de 45 anos para mais de 35 anos aumentaria a sensibilidade diagnóstica para os casos com IBM deste estudo de 83% para 100%. Com este estudo, foi possível caracterizar clínica e histológicamente pacientes com IBM em nosso meio, e fornecer indícios do benefício do uso de marcadores de degeneração e autofagia para o diagnóstico e para a determinação de vias ou sistemas celulares envolvidos na patogênese da doença / Sporadic inclusion body myositis (sIBM) is considered the most common acquired myopathy affecting adults aged over 50 years. Although included in the group of inflammatory myopathies, recent studies show a particular process of muscle degeneration characterized by abnormal deposit of protein aggregates in muscle fibers and abnormal operation of the main protein degradation systems. The aim of this study was to evaluate the clinical, histological and immunohistochemical patients with IBM. We evaluated 18 cases with IBM diagnostic of two of the main centers of neuromuscular diseases in Brazil (25 muscle biopsies). In an attempt to differentiate the IBM cases of other inflammatory myopathies, we determined the pattern of tissue expression of p-tau (p62), alfa-synuclein and TDP-43. Also evaluated the lysosomal function by acid phosphatase reaction (marking global lysosomal activity) and determining the markup for LC3B (autophagy marker). It was observed that IBM was predominant in males (61% of cases), white colored, with onset of clinical manifestations around 59 years old and the most common symptoms are muscle weakness, postural instability with high falls, dysphagia and weight loss, and may also present dyspnea. The diagnosis took an average of 7.4 years after the onset of symptoms and was often associated with the following comorbidities: hypertension, type 2 diabetes mellitus, osteopenia / osteoporosis, dyslipidemia and hyperuricemia / gout. The muscular damage pattern at IBM was characterized by tetraparesis predominantly proximal lower limbs and distal upper limbs. Serum creatine kinase levels in at least one of the measurements were elevated in all patients, but not exceeding 10 times normal. Immunosuppression was not effective in patients with IBM. The IBM histological findings included diversify dystrophic changes, endomysial inflammation, as well as the occurrence of rimmed vacuoles, in addition to high frequency of mitochondrial changes. Other characteristic muscle histological findings in IBM were increased lysosomal activity (overall increase in labeling for acid phosphatase), the presence of positive staining for beta-amyloid (intra-vacuolar by Congo red marking), increased muscle degradation (related to activation of LC3B, p-tau and p62 / SQSTM1) and muscle degeneration (marking for anti-phospo TDP-43 and ?-synuclein). Such changes have a high sensitivity and specificity. which makes these important complementary analyzes for accurate pathological diagnosis. We suggest that lowering the age of the onset of symptoms of greater than 45 years to older than 35 years would increase the diagnostic sensitivity for cases with IBM this study from 83% to 100%. With this study, it was possible to characterize clinically and histologically the patients with IBM in our centers, and provide evidence of the benefit of using degeneration and autophagy markers for diagnosis and for determining pathways or cellular systems involved in the pathogenesis of the disease