Browsing by Author "Vicente Miranda, Hugo"
Now showing 1 - 3 of 3
Results Per Page
Sort Options
- Glycation potentiates α-synuclein-associated neurodegeneration in synucleinopathiesPublication . Vicente Miranda, Hugo; Szego, Éva M.; Oliveira, Luís M.A.; Breda, Carlo; Darendelioglu, Ekrem; de Oliveira, Rita M.; Ferreira, Diana G.; Gomes, Marcos A.; Rott, Ruth; Oliveira, Márcia; Munari, Francesca; Enguita, Francisco J.; Simões, Tânia; Rodrigues, Eva F.; Heinrich, Michael; Martins, Ivo C.; Zamolo, Irina; Riess, Olaf; Cordeiro, Carlos; Ponces-Freire, Ana; Lashuel, Hilal A.; Santos, Nuno C.; Lopes, Luisa V.; Xiang, Wei; Jovin, Thomas M.; Penque, Deborah; Engelender, Simone; Zweckstetter, Markus; Klucken, Jochen; Giorgini, Flaviano; Quintas, Alexandre; Outeiro, Tiago F.α-Synuclein misfolding and aggregation is a hallmark in Parkinson's disease and in several other neurodegenerative diseases known as synucleinopathies. The toxic properties of α-synuclein are conserved from yeast to man, but the precise underpinnings of the cellular pathologies associated are still elusive, complicating the development of effective therapeutic strategies. Combining molecular genetics with target-based approaches, we established that glycation, an unavoidable age-associated post-translational modification, enhanced α-synuclein toxicity in vitro and in vivo, in Drosophila and in mice. Glycation affected primarily the N-terminal region of α-synuclein, reducing membrane binding, impaired the clearance of α-synuclein, and promoted the accumulation of toxic oligomers that impaired neuronal synaptic transmission. Strikingly, using glycation inhibitors, we demonstrated that normal clearance of α-synuclein was re-established, aggregation was reduced, and motor phenotypes in Drosophila were alleviated. Altogether, our study demonstrates glycation constitutes a novel drug target that can be explored in synucleinopathies as well as in other neurodegenerative conditions.
- Heat-mediated enrichment of α-synuclein from cells and tissue for assessing post-translational modificationsPublication . Vicente Miranda, Hugo; Xiang, Wei; Oliveira, Rita M. de; Simões, Tânia; Pimentel, José; Klucken, Jochen; Penque, Deborah; Outeiro, Tiagoα-synuclein (α-syn) is the major component of Lewy bodies, a pathological hallmark of Parkinson's disease and other synucleinopathies. The characterization of α-syn post-translational modifications (PTMs), thought to interfere with its aggregation propensity and cellular signaling, has been limited by the availability of extraction methods of endogenous protein from cells and tissues, and by the availability of antibodies toward α-syn PTMs. Here, by taking advantage of α-syn thermostability, we applied a method to achieve high enrichment of soluble α-syn both from cultured cells and brain tissues followed by proteomics analysis. Using this approach, we obtained 98% α-syn sequence coverage in a variety of model systems, including a transgenic mouse model of PD, and validated the strategy by identifying previously described PTMs such as phosphorylation and N-terminal acetylation. Our findings demonstrate that this procedure overcomes existing technical limitations and can be used to facilitate the systematic study of α-syn PTMs, thereby enabling the clarification of their role under physiological and pathological conditions. Ultimately, this approach may enable the development of novel biomarkers and strategies for therapeutic intervention in synucleinopathies. In this study, we describe a method for enriching alpha-synuclein (α-syn) from a variety of biological samples, from cultured cells to brain tissues. Enrichment of α-syn was achieved by heating samples, further facilitating the identification of specific post-translational modifications by immunoblot, or mass spectrometry-based techniques. This approach will contribute to the clarification of the role of α-syn PTMs in Parkinson's disease.
- The mechanism of sirtuin 2-mediated exacerbation of alpha-synuclein toxicity in models of Parkinson diseasePublication . de Oliveira, Rita Machado; Vicente Miranda, Hugo; Francelle, Laetitia; Pinho, Raquel; Szegö, Éva M.; Martinho, Renato; Munari, Francesca; Lázaro, Diana F.; Moniot, Sébastien; Guerreiro, Patrícia; Fonseca-Ornelas, Luis; Marijanovic, Zrinka; Antas, Pedro; Gerhardt, Ellen; Enguita, Francisco Javier; Fauvet, Bruno; Penque, Deborah; Pais, Teresa Faria; Tong, Qiang; Becker, Stefan; Kügler, Sebastian; Lashuel, Hilal Ahmed; Steegborn, Clemens; Zweckstetter, Markus; Outeiro, Tiago FlemingSirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.