Author(s): Barbosa, Marta Isabel da Silva Rodrigues
Date: 2013
Persistent ID: http://hdl.handle.net/10362/10925
Origin: Repositório Institucional da UNL
Subject(s): Inflammatory mediators; Microglia modulatory effects of motoneuron degeneration; Microglia-motoneuron communication; Neuroinflammation; SOD1 accumulation
Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina
Convergence of pathways in motoneuron (MN) injury include microglia in the initiation and progression of Amyotrophic Lateral Sclerosis (ALS). Neuroinflammation is a pathological hallmark of ALS and microglia may acquire neurotoxic or neuroprotective properties in response to misfolded superoxide dismutase-1 (SOD1) or other molecules produced by the injured MN. We assessed: (i) the role of microglia in preventing/restoring MN dysfunction using a mixed culture of NSC-34 MN-like cells (mutated in G93A) and of N9 microglia cells, added at 0 or 2 days-invitro(M0, M2) and cultured till 4 and 7 days-in-vitro; (ii) neurodegenerative network in organotypic cultures from lumbar segments of spinal cord (SC) obtained from the ALS mice model TgSOD1-G93A at 7 day-old and aged for 10 days-in-vitro, as well as the response to lipopolysaccharide (LPS, 1μg/mL) immunostimulation. Western blot assays for SOD1, high-mobility-group-box-protein-1 (HMGB1) and toll-like receptor-4 (TLR-4), and fluorimetric/colorimetric assays for ATP, glutamate and nitric oxide (NO), were used. Microglia (M0/M2) decreased the accumulation of human/mouse mutated SOD1 (P<0.01). In addition, elevation of glutamate efflux (P<0.01), and reduction of extracellular ATP (P<0.01), MMP-2(P<0.05) and MMP-9 (P<0.01) was observed by M2 at 7 days-in-vitro. Reduction of NO (P<0.05) and MMP-2 (P<0.01) was obtained with M0. HMGB1 increased by M0 and decreased by M2, suggesting HMGB1 release from the cell. Accumulation of SOD1 was verified in SC organotypic cultures, but no changes in ATP or NO were obtained, although a slight decrease in ATP by LPS was verified. Downregulation of TLR-4 by LPS may indicate the exhaustion of the inflammatory response mechanisms in the aged SC culture. Together, these results suggest that microglia by inhibiting MMP activation and HMGB1 cytoplasmic translocation in the ALS model are key in modulating MN degeneration and should be considered as therapeutic targets in ALS.
supported by FEDER (COMPETE Programme) and by National funds (Fundação para a Ciência e a Tecnologia – FCT, Portugal) with the projects PTDC/SAU-FAR/118787/2010 to D.B. and PEst-OE/SAU/UI4013/2011 and 2012 to iMed.UL
