Effect of psychosine on inducible nitric-oxide synthase expression under different culture conditions: implications for Krabbe disease.

BACKGROUND AND OBJECTIVES Krabbe disease is a neuro-inflammatory disorder in which galactosyl sphingosine (psychosine) accumulates in nervous tissues. Despite some leads in elucidating the mechanism of psychosine action on different cells of brain, there still remain gaps in the knowledge and mechanisms behind events in the pathogenic cascade of Krabbe disease. Inflammation in the brain in Krabbe disorder is an important factor in neural damage. This study was undertaken to access the role of psychosine in the regulation of nitric oxide (NO) and inducible nitric oxide synthase (iNOS), i.e., inflammatory markers, under two different conditions viz., using a single cell line and using primary mixed glial cells. MATERIALS AND METHODS BV2 murine microglial cells and murine primary mixed glial cells were used during this study. The cell lines, after 12 hr serum starvation, were treated with lipopolysaccharide (LPS) at 25 ng/ml in the absence or presence of increasing concentrations of psychosine (5, 10 and 15 microM). Formation of NO was estimated using Greiss reagent, and expression of iNOS was done by SDS-page followed by western blotting using anti-iNOS antibody. RESULTS In BV2 cells it was found that LPS (25 ng/ml) treatment, induced production of NO, but the LPS induced NO production was inhibited when LPS was used in combination with psychosine. This result was corroborated by parallel trend seen in iNOS expression under same conditions. Contrary to this, LPS (25 ng/ml) induced production of NO in primary mixed glial cells was dose dependently enhanced when LPS was used in combination with psychosine (5, 10 and 15 microM). This result was also corroborated by iNOS expression under same experimental conditions. DISCUSSION This study suggests that in-vitro data obtained using individual cell lines may not reflect the actual complex intricacies involved in development of Krabbe brain pathology. And that the effect of psychosine in Krabbe brain may be modulated by presence of LPS or other pro-inflammatory stimuli in the brain of these children, e.g., after an infection.