PIG-A mutations in normal hematopoiesis

Paroxysmal nocturnal hemoglobinuria (PNH) is caused by phosphatidylinositol glycan–class A (PIG-A) mutations in hematopoietic stem cells (HSCs). PIG-A mutations have been found in granulocytes from most healthy individuals, suggesting that these spontaneous PIG-A mutations are important in the pathogenesis of PNH. It remains unclear if these PIG-A mutations have relevance to those found in PNH. We isolated CD34+ progenitors from 4 patients with PNH and 27 controls. The frequency of PIG-A mutant progenitors was determined by assaying for colony-forming cells (CFCs) in methylcellulose containing toxic doses of aerolysin (1 × 10-9 M). Glycosylphosphatidylinositol (GPI)–anchored proteins serve as receptors for aerolysin; thus, PNH cells are resistant to aerolysin. The frequency of aerolysin resistant CFC was 14.7 ± 4.0 × 10-6 in the bone marrow of healthy donors and was 57.0 ± 6.7 × 10-6 from mobilized peripheral blood. DNA was extracted from individual day-14 aerolysin-resistant CFCs and the PIG-A gene was sequenced to determine clonality. Aerolysin-resistant CFCs from patients with PNH exhibited clonal PIG-A mutations. In contrast, PIG-A mutations in the CFCs from controls were polyclonal, and did not involve T cells. Our data confirm the finding that PIG-A mutations are relatively common in normal hematopoiesis; however, the finding suggests that these mutations occur in differentiated progenitors rather than HSCs

Manganese Superoxide Dismutase Protects nNOS Neurons from NMDA and Nitric Oxide-Mediated Neurotoxicity

Neuronal nitric oxide synthase (nNOS) neurons kill adjacent neurons through the action of NMDA-glutamate receptor activation, although they remain relatively resistant to the toxic effects of NMDA and NO. The molecular basis of the resistance of nNOS neurons to toxic insults is unknown. To begin to understand the molecular mechanisms of the resistance of nNOS neurons, we developed a pheochromacytoma-derived cell line (PC12) that is resistant to the toxic effects of NO. We found through serial analysis of gene expression (SAGE) that manganese superoxide dismutase (MnSOD) is enriched in the NO-resistant PC12 cell-derived line (PC12-R). Antisense MnSOD renders PC12-R cells sensitive to NO toxicity and increases the sensitivity to NO in the parental, NO-sensitive PC12 line (PC12-S). Adenoviral transfer of MnSOD protects PC12-S cells against NO toxicity. We extended these studies to cortical cultures and showed that MnSOD is enriched in nNOS neurons and that antisense MnSOD renders nNOS neurons susceptible to NMDA neurotoxicity, although it has little effect on the overall susceptibility of cortical neurons to NMDA toxicity. Overexpression of MnSOD provides dramatic protection against NMDA and NO toxicity in cortical cultures, but not against kainate or AMPA neurotoxicity. Furthermore, nNOS neurons from MnSOD(-/-) mice are markedly sensitive to NMDA toxicity. Adenoviral transfer of MnSOD to MnSOD(-/-) cultures restores resistance of nNOS neurons to NMDA toxicity. Thus, MnSOD is a major protective protein that appears to be essential for the resistance of nNOS neurons in cortical cultures to NMDA mediated neurotoxicity