Expression and Purification of Quinine Dihydro Pteridine Reductase from astrocytes and its significance in the astrocyte pathology.

Quinine dihydropteridinereductase (QDPR) is involved in the synthesis of tetradihydrobiopteridine (BH4) that serve as cofactor for many aromatic hydroxylases including induced nitric oxide synthase (NOS) leading to NO production. Increased activity of QDPR has been associated with decrease levels of TGF-β, a cytokine that regulates the immune response and that elevated levels of NO has been associated with neurodegenerative diseases. Thus, expression of QDPR in astrocytes is essential to study the pathological changes observed in many neurodegenerative disorders. We have expressed QDPR in astrocytes and generated stably expressing clones that overexpresses QDPR. We further verified the specificity of QDPR expression using immunofluorescence and immunoblotting. To further confirm, we purified QDPR using Ni-NTA column and subjected the purified fraction to immunoblotting using anti-QDPR antibody and identified two major protein products of QDPR resolving at 25 and 17 kDa as reported in the literature. In order to further assess the significance of QDPR expression, we verified the expression of iNOS in QDPR over expressing cells. We show for the first time statistically significant up regulation of iNOS in QDPR overexpressing astrocytes. Increased expression of iNOS associated with astrocyte pathology seen in many neurodegenerative disorders may have implications in autoimmune neurodegenerative disorders.