Structural Characterization of Heparin-induced GAPDH Protofibrils Preventing α-synuclein Oligomeric Species Toxicity

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional enzyme that has been associated with neurodegenerative diseases. GAPDH colocalizes with α-synuclein in amyloid aggregates in post-mortem tissue of patients with sporadic Parkinson disease and promotes the formation of Lewy body-like inclusions in cell culture. In a previous work, we showed that glycosaminoglycan-induced GAPDH prefibrillar species accelerate the conversion of α-synuclein to fibrils. However, it remains to be determined whether the interplay among glycosaminoglycans, GAPDH, and α-synuclein has a role in pathological states. Here, we demonstrate that the toxic effect exerted by α-synuclein oligomers in dopaminergic cell culture is abolished in the presence of GAPDH prefibrillar species. Structural analysis of prefibrillar GAPDH performed by small angle x-ray scattering showed a particle compatible with a protofibril. This protofibril is shaped as a cylinder 22 nm long and a cross-section diameter of 12 nm. Using biocomputational techniques, we obtained the first all-atom model of the GAPDH protofibril, which was validated by cross-linking coupled to mass spectrometry experiments. Because GAPDH can be secreted outside the cell where glycosaminoglycans are present, it seems plausible that GAPDH protofibrils could be assembled in the extracellular space kidnapping α-synuclein toxic oligomers. Thus, the role of GAPDH protofibrils in neuronal proteostasis must be considered. The data reported here could open alternative ways in the development of therapeutic strategies against synucleinopathies like Parkinson disease.Fil: Avila, Cesar Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Torres Bugeau, Clarisa Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; ArgentinaFil: Barbosa, Leandro R. S.. Universidade de Sao Paulo; BrasilFil: Morandé Sales, Elisa. Universidade de Sao Paulo; BrasilFil: Ouidja, Mohand O.. Inserm; Francia. Centre National de la Recherche Scientifique; FranciaFil: Socias, Sergio Benjamin. Inserm; FranciaFil: Celej, Maria Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Química Biológica de Córdoba (p); Argentina. Universidad Nacional de Cordoba. Facultad de Ciencias Quimicas; ArgentinaFil: Raisman Vozari, Rita. Universidade de Sao Paulo; BrasilFil: Papy Garcia, Dulce. Centre National de la Recherche Scientifique; FranciaFil: Itri, Rosangela. Universidade de Sao Paulo; BrasilFil: Chehin, Rosana Nieves. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia. Instituto de Química Biológica; Argentin

Doxycycline Suppresses Microglial Activation by Inhibiting the p38 MAPK and NF-kB Signaling Pathways

In neurodegenerative diseases, the inflammatory response is mediated by activated glial cells, mainly microglia, which are the resident immune cells of the central nervous system. Activated microglial cells release proinflammatory mediators and neurotoxic factors that are suspected to cause or exacerbate these diseases. We recently demonstrated that doxycycline protects substantia nigra dopaminergic neurons in an animal model of Parkinson’s disease. This effect was associated with a reduction of microglial cell activation, which suggests that doxycycline may operate primarily as an anti-inflammatory drug. In the present study, we assessed the anti-inflammatory potential of doxycycline using lipopolysaccharide (LPS)-activated primary microglial cells in culture as a model of neuroinflammation. Doxycycline attenuated the expression of key activation markers in LPS-treated microglial cultures in a concentration-dependent manner. More specifically, doxycycline treatment lowered the expression of the microglial activation marker IBA-1 as well as the production of ROS, NO, and proinflammatory cytokines (TNF-α and IL-1β). In primary microglial cells, we also found that doxycycline inhibits LPS-induced p38 MAP kinase phosphorylation and NF-kB nuclear translocation. The present results indicate that the effect of doxycycline on LPS-induced microglial activation probably occurs via the modulation of p38 MAP kinase and NF-kB signaling pathways. These results support the idea that doxycycline may be useful in preventing or slowing the progression of PD and other neurodegenerative diseases that exhibit altered glia function.Fil: Santa Cecília, Flávia V.. Universite Paris Sorbonne - Paris Iv; . Universidade de Sao Paulo; BrasilFil: Socias, Sergio Benjamin. Universite Paris Sorbonne - Paris Iv; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ouidja, Mohand O.. Universite Paris Sorbonne - Paris Iv; FranciaFil: Sepulveda Diaz, Julia E.. Universite Paris Sorbonne - Paris Iv; FranciaFil: Acuña, Leonardo. Universite Paris Sorbonne - Paris Iv; Francia. 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; ArgentinaFil: Silva, Rangel L.. Universidade de Sao Paulo; BrasilFil: Michel, Patrick P.. Universite Paris Sorbonne - Paris Iv; FranciaFil: Del Bel, Elaine. Universidade de Sao Paulo; BrasilFil: Cunha, Thiago M.. Universidade de Sao Paulo; BrasilFil: Raisman Vozari, Rita. Universite Paris Sorbonne - Paris Iv; Franci