Caracterización estructural de oligómeros amiloides de α-sinucleína y su interacción con membranas modelo

α-sinucleína (AS) es una proteína expresada mayormente en cerebro y su función está asociada a la dinámica de vesículas sinápticas en neuronas dopaminérgicas. Su agregación anómala amiloide está vinculada con numerosas patologías neurodegenerativas como por ejemplo la enfermedad de Parkinson. A pesar de los grandes avances realizados, aún se desconocen cuáles son los mecanismos patogénicos asociados a estas enfermedades. Los oligómeros amiloides pre-fibrilares son señalados como las especies más neurotóxicas. Al respecto, la hipótesis mayormente aceptada sugiere una ganancia de toxicidad de AS cuando se encuentra en estado oligomérico. Sin embargo, una pérdida de la función de la proteína al autoensamblarse en estos agregados podría también contribuir a la disfunción celular. En esta tesis se dirigieron los esfuerzos en estos dos sentidos, por un lado, aportando información estructural de las especies oligoméricas para comprender las bases moleculares de su toxicidad y por el otro, evaluando el impacto de la oligomerización en las propiedades de unión a membranas lipídicas, claves para la función de la proteína. Utilizando técnicas espectroscópicas se logró obtener información sobre el arreglo supramolecular de una población de especies oligoméricas de AS. Este ensamble resultó estar formado por agregados estructurados, ricos en estructura hoja-β antiparalela que poseen un patrón bien definido de interacciones intermoleculares. Se demostró que esta estructura distintiva también está presente en especies que se pueblan a lo largo del proceso de agregación amiloide, evidenciando además que este proceso involucra un marcado rearreglo conformacional de la proteína. A su vez, se midió cuantitativamente la unión de AS en su estado monomérico y oligomérico a membranas de distinta composición y curvatura. Se evidenció que la oligomerización de la proteína influye marcadamente en su interacción con membranas, cambiando su sensibilidad a curvatura y alterando su afinidad, llegando en algunos casos a abolir su asociación completamente. La información estructural obtenida en este trabajo, junto con el estudio de unión a biomembranas, constituyen un avance clave para la comprensión de la toxicidad y pérdida de la función de AS asociada a las patologías neurodegenerativas.α-synuclein (AS) is a protein highly expressed in the brain and its function is linked to synaptic vesicle dynamics in dopaminergic neurons. Its aberrant amyloid aggregation is associated with several neurodegenerative disorders such as Parkinson's disease. Despite the progress that has been made over the years, the pathogenic mechanisms governing these diseases remain unknown. Prefibrillar amyloid oligomeric species are pointed as the more neurotoxic species. In this regard, the prevailing hypothesis indicates a gain-of-toxicity mechanism when the protein is in its oligomeric state. Nevertheless, a loss-of-function due to protein self-assembling might also be involved in cellular dysfunction. In this thesis, we directed our efforts to pave the molecular understanding of these two possibilities. On one hand, providing structural information on oligomeric species to understand the molecular basis of their toxicity and on the other, evaluating the impact of AS oligomerization on its membrane lipid binding properties, key elements for protein function. By means of spectroscopic techniques we gathered information on the supramolecular arrangement of a population of oligomeric species. This ensemble was formed by structured, antiparallel β-sheet rich aggregates with a well-defined pattern of intermolecular contacts. We demonstrated that this distinctive structural feature is also present at the level of prefibrillar intermediates in the pathway of amyloid formation, also showing that this process involves a large conformational re-arrangement of the protein. Simultaneously, we quantitatively measured the binding of AS in its monomeric and oligomeric state to membranes of different curvature and composition. We showed that protein oligomerization has a deep impact on its interaction with membranes, changing its curvature sensitivity and modifying its affinity at such an extent that binding is abolished in some cases. The structural information attained in this work, together with the membrane binding study, constitute a key advance for the understanding of toxicity and loss-of-function mechanisms of AS associated to neurodegenerative diseases.Fil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

From Work Well-Being to Burnout: A Hypothetical Phase Model

Upon exposure to chronic stressors, how do individuals move from being in a healthy state to a burnout? Strikingly in literature, this has prevailed a categorical view rather than a dimensional one, thus the underlying process that explains the transition from one state to another remains unclear. The aims of the present study are (a) to examine intermediate states between work engagement and burnout using cluster analysis and (b) to examine cortisol differences across these states. Two-hundred and eighty-one Argentine workers completed self-report measures of work engagement and burnout. Salivary cortisol was measured at three time-points: immediately after awakening and 30 and 40min thereafter. Results showed four different states based on the scores in cynicism, exhaustion, vigor, and dedication: engaged, strained, cynical, and burned-out. Cortisol levels were found to be moderate in the engaged state, increased in the strained and cynical states, and decreased in the burned-out state. The increase/decrease in cortisol across the four stages reconciles apparent contradictory findings regarding hypercortisolism and hypocortisolism, and suggests that they may represent different phases in the transition from engagement to burnout. A phase model from engagement to burnout is proposed and future research aimed at evaluating this model is suggested.Fil: Morera, Luis Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Siglo 21; ArgentinaFil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Siglo 21; ArgentinaFil: Trógolo, Mario Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Siglo 21; ArgentinaFil: Guido, Mario Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Medrano, L. A.. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Siglo 21; Argentin

Amyloid oligomerization of the Parkinson's disease related protein α-synuclein impacts on its curvature-membrane sensitivity

The amyloid aggregation of the presynaptic protein α-synuclein (AS) is pathognomonic of Parkinson's disease and other neurodegenerative disorders. Physiologically, AS contributes to synaptic homeostasis by participating in vesicle maintenance, trafficking, and release. Its avidity for highly curved acidic membranes has been related to the distinct chemistry of the N-terminal amphipathic helix adopted upon binding to appropriated lipid interfaces. Pathologically, AS populate a myriad of toxic aggregates ranging from soluble oligomers to insoluble amyloid fibrils. Different gain-of-toxic function mechanisms are linked to prefibrillar oligomers which are considered as the most neurotoxic species. Here, we investigated if amyloid oligomerization could hamper AS function as a membrane curvature sensor. We used fluorescence correlation spectroscopy to quantitatively evaluate the interaction of oligomeric species, produced using a popular method based on lyophilization and rehydration, to lipid vesicles of different curvatures and compositions. We found that AS oligomerization has a profound impact on protein-lipid interaction, altering binding affinity and/or curvature sensitivity depending on membrane composition. Our work provides novel insights into how the formation of prefibrillar intermediate species could contribute to neurodegeneration due to a loss-of-function mechanism.Fil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Ambroggio, Ernesto Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Vilcaes, Aldo Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: James, Nicholas G.. University of Hawaii at Manoa; Estados UnidosFil: Jameson, David M.. University of Hawaii at Manoa; Estados UnidosFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Autophagy down regulates pro-inflammatory mediators in BV2 microglial cells and rescues both LPS and alpha-synuclein induced neuronal cell death

Autophagy is a fundamental cellular homeostatic mechanism, whereby cells autodigest parts of their cytoplasm for removal or turnover. Neurodegenerative disorders are associated with autophagy dysregulation, and drugs modulating autophagy have been successful in several animal models. Microglial cells are phagocytes in the central nervous system (CNS) that become activated in pathological conditions and determine the fate of other neural cells. Here, we studied the effects of autophagy on the production of pro-inflammatory molecules in microglial cells and their effects on neuronal cells. We observed that both trehalose and rapamycin activate autophagy in BV2 microglial cells and down-regulate the production of pro-inflammatory cytokines and nitric oxide (NO), in response to LPS and alpha-synuclein. Autophagy also modulated the phosphorylation of p38 and ERK1/2 MAPKs in BV2 cells, which was required for NO production. These actions of autophagy modified the impact of microglial activation on neuronal cells, leading to suppression of neurotoxicity. Our results demonstrate a novel role for autophagy in the regulation of microglial cell activation and pro-inflammatory molecule secretion, which may be important for the control of inflammatory responses in the CNS and neurotoxicity.Fil: Bussi, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Peralta Ramos, Javier María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Arroyo, Daniela Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Gaviglio, Emilia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; ArgentinaFil: Wang, Ji Ming. National Cancer Institute at Frederick; Estados UnidosFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; ArgentinaFil: Iribarren, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin

Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.Fil: Bussi, Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Peralta Ramos, Javier María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Arroyo, Daniela Soledad. Universidad Nacional de Córdoba; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina. Universidad Nacional de Córdoba; ArgentinaFil: Ronchi, Paolo. European Molecular Biology Laboratory; AlemaniaFil: Kolovou, Androniki. European Molecular Biology Laboratory; AlemaniaFil: Wang, Ji M.. National Cancer Institute at Frederick; Estados UnidosFil: Florey, Oliver. Babraham Institute; Reino UnidoFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Schwab, Yannick. European Molecular Biology Laboratory; AlemaniaFil: Ktistakis, Nicholas. Babraham Institute; Reino UnidoFil: Iribarren, Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba; Argentin

Structural Insights into Amyloid Oligomers of the Parkinson Disease-related Protein α-Synuclein

The presence of intraneuronal deposits mainly formed by amyloid fibrils of the presynaptic protein α-synuclein (AS) is a hallmark of Parkinson disease. Currently, neurotoxicity is attributed to prefibrillar oligomeric species rather than the insoluble aggregates, although their mechanisms of toxicity remain elusive. Structural details of the supramolecular organization of AS oligomers are critically needed to decipher the structure-toxicity relationship underlying their pathogenicity. In this study, we employed site-specific fluorescence to get a deeper insight into the internal architecture of AS oligomeric intermediates. We demonstrate that AS oligomers are ordered assemblies possessing a well defined pattern of intermolecular contacts. Some of these contacts involve regions that form the β-sheet core in the fibrillar state, although their spatial arrangement may differ in the two aggregated forms. However, even though the two termini are excluded from the fibrillar core, they are engaged in a number of intermolecular interactions within the oligomer. Therefore, substantial structural remodeling of early oligomeric interactions is essential for fibril growth. The intermolecular contacts identified in AS oligomers can serve as targets for the rational design of anti-amyloid compounds directed at preventing oligomeric interactions/reorganizations.Fil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Structural remodeling during amyloidogenesis of physiological Nα-acetylated α-synuclein

The misfolding and aggregation of the presynaptic protein α-synuclein (AS) into amyloid fibrils is pathognomonic of Parkinson's disease, though the mechanism by which this structural conversion occurs is largely unknown. Soluble oligomeric species that accumulate as intermediates in the process of fibril formation are thought to be highly cytotoxic. Recent studies indicate that oligomer-to-fibril AS transition plays a key role in cell toxicity and progression of neurodegeneration. We previously demonstrated that a subgroup of oligomeric AS species are ordered assemblies possessing a well-defined pattern of intermolecular contacts which are arranged into a distinctive antiparallel β-sheet structure, as opposed to the parallel fibrillar fold. Recently, it was demonstrated that the physiological form of AS is N-terminally acetylated (Ac-AS). Here, we first showed that well-characterized conformational ensembles of Ac-AS, namely monomers, oligomers and fibrils, recapitulate many biophysical features of the nonacetylated protein, such as hydrodynamic, tinctorial, structural and membrane-leakage properties. Then, we relied on ATR-FTIR spectroscopy to explore the structural reorganization during Ac-AS fibrillogenesis. We found that antiparallel β-sheet transient intermediates are built-up at early stages of aggregation, which then evolve to parallel β-sheet fibrils through helix-rich/disordered species. The results are discussed in terms of regions of the protein that might participate in this structural rearrangement. Our work provides new insights into the complex conformational reorganization occurring during Ac-AS amyloid formation.Fil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Sarroukh, Rabia. Université Libre de Bruxelles; BélgicaFil: Yunes Quartino, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; ArgentinaFil: Ruysschaert, Jean-Marie. Université Libre de Bruxelles; BélgicaFil: Raussens, Vincent. Université Libre de Bruxelles; BélgicaFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentin

Neuronal inclusions of alpha-synuclein contribute to the pathogenesis of Krabbe disease

Demyelination is a major contributor to the general decay of neural functions in children with Krabbe disease. However, recent reports have indicated a significant involvement of neurons and axons in the neuropathology of the disease. In this study, we have investigated the nature of cellular inclusions in the Krabbe brain. Brain samples from the twitcher mouse model for Krabbe disease and from patients affected with the infantile and late-onset forms of the disease were examined for the presence of neuronal inclusions. Our experiments demonstrated the presence of cytoplasmic aggregates of thioflavin-S-reactive material in both human and murine mutant brains. Most of these inclusions were associated with neurons. A few inclusions were detected to be associated with microglia and none were associated with astrocytes or oligodendrocytes. Thioflavin-S-reactive inclusions increased in abundance, paralleling the development of neurological symptoms, and distributed throughout the twitcher brain in areas of major involvement in cognition and motor functions. Electron microscopy confirmed the presence of aggregates of stereotypic β-sheet folded proteinaceous material. Immunochemical analyses identified the presence of aggregated forms of α-synuclein and ubiquitin, proteins involved in the formation of Lewy bodies in Parkinson's disease and other neurodegenerative conditions. In vitro assays demonstrated that psychosine, the neurotoxic sphingolipid accumulated in Krabbe disease, accelerated the fibrillization of α-synuclein. This study demonstrates the occurrence of neuronal deposits of fibrillized proteins including α-synuclein, identifying Krabbe disease as a new α-synucleinopathy.Fil: Smith, Benjamin R.. University of Illinois; Estados UnidosFil: Santos, Marta B.. University of Illinois; Estados UnidosFil: Marshal, Michael S.. University of Illinois; Estados UnidosFil: Cantuti Castelvetri, Ludovico. University of Illinois; Estados UnidosFil: Lopez Rosas, Aurora. University of Illinois; Estados UnidosFil: Li, Guannan. University of Illinois; Estados UnidosFil: Van Breemen, Richard B.. University of Illinois; Estados UnidosFil: Claycomb, Kumiko I.. University Of Connecticut; Estados UnidosFil: Gallea, Jose Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones en Química Biológica de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Centro de Investigaciones en Química Biológica de Córdoba; ArgentinaFil: Crocker, Stephen. University Of Connecticut; Estados UnidosFil: Givogri, Maria I.. University of Illinois; Estados UnidosFil: Bongarzone, Ernesto R.. University of Illinois; Estados Unido