FP6 Project NEUROIMAGE

Transcription

1 LOGO FP6 Project NEUROIMAGE Ivan Spasojević Centre for Laser Microscopy

2 Lecturer information Ivan Spasojević Phone: Present: Postdoc at the CLM Performing research at the Institute for Multidisciplinary Research as Assistant Prof. E. & Q.: 2002 Molecular biology and physiology BSc Faculty of Biology, University of Belgrade 2006 Biohysics PhD University of Belgrade Postdoc CLM

3 Protective role of fructose in the metabolism of C6 astroglioma cells exposed to hydrogen peroxide Focus of my research: Oxidative metabolism (17 out of 20 of my scientific publications (original papers and reviews) are on O.M.) Various pathophysiologies (neurodegeneration, malignancies, autoimmune diseases ) are related to oxidative stress (increased level of reactive species) Antioxidative therapy generally ineffective Refractory response maintains responsive redox poise, thus enabling participation of reactive species (e.g. H 2 O 2 ) in signaling How to help the organism to fight oxidative stress against its own will We proposed that, in contrast to supplemented antioxidants, the organism will not reject the energy supply if presented in times of crisis Antioxidative activities of sugars against H 2 O 2 related oxidation were compared using EPR spin-trapping spectroscopy Rank of order: F16BP>F1P>F6P>fructose>glucose=mannitol (Spasojevic et al. Carbohyd. Res. 2009, 344, )

4 Fructose con & pro Chronic application of fructose has negative consequences in Western societies may be a potentially important factor in the growing rates of obesity and the metabolic syndrome, as well as the notion that fructose intake is independently associated with an increased risk of kidney stone formation. Acute application showed numerous positive effects: Fructose protects tissues from anoxia and hypoxia F16BP prevents reperfusion injury, protects against septic shock, and provides protection against the cell damage associated with mth poisons and prooxidants F16BP is used to protect organs prepared for transplantation Fructose increases intracellular glutathione pool F16BP has anticonvulsant activity, and it is used in infusion of ischemic patients Fructose and F16BP pass BBB Fructose is transported into cells of nervous tissue by GLUT5 i GLUT2 F16BP is transported (passively and actively by dicarboxylate transporters) into cells of nervous tissue

5 Points on neurodegeneration Increased production of H 2 O 2 via different pathways Neurons are susceptible to oxidative damage low antioxidant level and regenerative capacity, specific geometry, high oxygen consumption Astroglia the main line of defense against oxidative stress in nervous tissue Of high importance is to help and protect astroglial cells

6 Experimental set up Antioxidative effects of fructose and its phosphorylated (intracellular) forms, against H 2 O 2, were compared to glucose Model system: C6 astroglioma cells Initiator of oxidative stress: H 2 O 2 (3 mm pathophysiological level in vivo) for 10 minutes and than washed Cell media: glucose (10 mm), fructose (10 mm), glucose + fructose (5 + 5 mm), glucose (10 mm) + F16BP/F1P/F6P (5 mm) Methods: Confocal microscopy with fluorescent mitochondria markers and MTT assay Parameters: Relative oxidative status, Viability

7 MTO fluorescence in C6 cells upon exposure to H 2 O 2 depends on hexose supplementation Mitotracker Orange -H 2 O 2 dependant rise in fluorescence intensity - Used for evaluating oxidative status

8 Viability (white) and oxidative status (grey) of C6 cells exposed to H 2 O 2 in media with different infusion sugars Mean per pixel intensity/5 experimental days/5 images per dish Values are presented relative to controls with no H 2 O 2. The viability and oxidative status of untreated cells were 1±0.07 and 1±0.13. Statistical significance (p<0.05) relative to cells treated in glucose (black star) or relative to control (white). Significantly higher viability was obtained in the medium containing equal amounts of fructose and glucose

9 FDA/PI assay Green (FDA) alive cells Red (PI) dead cells Morphological changes are obvious (greater optical thickness) -H 2 O 2 provokes aggregation of F actin microfilaments

10 Protective effects of phosphorylated fructose In vivo concentration of F16BP in brain tissue of rats given intraperitoneal F16BP (0.5 g/kg) ~ 2mM

11 Protective effects of phosphorylated fructose Mean per pixel intensity/5 experimental days/5 images per dish Values are presented relative to controls with no H 2 O 2. Statistical significance (p<0.05) relative to controls (black star) and relative to cells treated in glucose (white).

12 Discussion Fructose and F16BP are the most protective Protective effects are observed at concentrations corresponding to in vivo levels Fructose is transported into the cells where it provides antioxidative protection directly and indirectly via its intracellular forms The antioxidative potential of fructose is directly related to C6 cell survival (MTT) Results concur with data obtained in EPR study Both pass BBB F16BP is transported (passively and actively by dicarboxylate transporters) into cells of nervous tissue Fructose and F16BP have already showed protective effects in pathophysiologies related to disturbed oxidative status F16BP level in brain tissue remains high long after it has fallen in peripheral tissues Fructose and F16BP could be used in cytoprotective therapy of neurodegenerative diseases Spasojevic et al. Carbohyd. Res. 2009, in press doi: /j.carres

13 Future studies Protective effects of fructose and F16BP in other cultured cells as model systems (astrocytes, neurones) In vivo studies of protective effects of fructose and F16BP on models of neurodiseases related to oxidative stress

14 Center for Laser Microscopy, School of Biology, University of Belgrade LOGO Ivan Spasojević, Aleksandar Bajić, Katarina Jovanović, Prof. Pavle Andjus Institute for Biological Research Sinisa Stankovic, Belgrade Prof. Mihajlo Spasić