, 2010). On the other hand, many studies suggest a neuroprotective role for GM1 in several disease models (Krajnc et al., 1994, Lazzaro et al., 1994, Augustinsson et al., 1997, Svennerholm et al., 2002 and Sokolova et al., 2007). Several studies have addressed a pivotal role for GSK3β signaling pathway in neuronal death
and disease development observed in Alzheimer’s (Hooper et al., 2008, Hernández et al., 2009a and Hernández et al., 2009b). An amyloid induced activation (dephosphorylation) of GSK3β has been shown in some experimental models, and a correlation between its activity and the neurotoxicity triggered by this peptide. Koh et al. (2008) proposed the analysis of GSK3β phosphorylation as a biochemical parameter in the investigation of possible neuroprotective drugs. Organotypic hippocampal slice cultures are a considerable alternative to animal model experiments. GW-572016 in vivo Cultured slices maintain the cell architecture selleck products and interneuronal connections, allowing for a long in vitro survival period ( Stoppini et al., 1991 and Tavares et al., 2001). They have been used to
investigate molecular mechanisms involved in cytotoxicity, such as the ones that are determined by oxygen and glucose deprivation ( Valentim et al., 2003, Cimarosti et al., 2005, Zamin et al., 2006, Horn et al., 2005 and Horn et al., 2009) and Aβ toxicity ( Ito et al., 2003, Nassif et al., 2007 and Frozza et al., 2009). This methodology has also been used for DNA ligase neuroprotection
strategy evaluations ( Cimarosti et al., 2006, Simão et al., 2009, Bernardi et al., 2010 and Hoppe et al., 2010). The aim of this study was to examine the effect of Aβ treatment to organotypic hippocampal slice cultures on ganglioside expression, as well as the GM1 effect on Aβ-induced toxicity, as assessed by cellular death and GSK3β phosphorylation. Acrylamide, bisacrylamide, SDS and β-mercaptoethanol used in sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS–PAGE) were obtained from Sigma (St. Louis, MO, USA) as well as Aβ25–35, Aβ35–25, propidium iodide (PI), standard glycolipids and the ganglioside GM1 used in culture incubation. Polyclonal antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-rabbit IgG peroxidase-conjugated and reagents to detect chemiluminescence (ECL) were purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA). Millicell culture inserts (Millicell®-CM, 0.4 μm) were obtained from Millipore (Millipore®, Bedford, MA, USA), 6-well culture plates were from TPP (Tissue culture test plates TPP®, Switzerland). Culture medium, HBSS, fungizone and heat inactivated horse serum were obtained from GIBCO (Grand Island, NY, USA). Gentamicin was from Schering–Plough (Rio de Janeiro, Brazil). D-[1-C14] galactose (57 mCi/mmol) was obtained from Amersham Life Science (Buckinghamshire, UK). Silicagel high performance thin layer chromatography (HPTLC) plates were supplied by Merck (Darmstadt, Germany).