Activation of microglial NADPH oxidase is synergistic with glial iNOS expression in inducing neuronal death: a dual-key mechanism of inflammatory neurodegeneration

BACKGROUND: Inflammation-activated glia are seen in many CNS pathologies and may kill neurons through the release of cytotoxic mediators, such as nitric oxide from inducible NO synthase (iNOS), and possibly superoxide from NADPH oxidase (NOX). We set out to determine the relative role of these species in inducing neuronal death, and to test the dual-key hypothesis that the production of both species simultaneously is required for significant neuronal death. METHODS: Primary co-cultures of cerebellar granule neurons and glia from rats were used to investigate the effect of NO (from iNOS, following lipopolysaccharide (LPS) and/or cytokine addition) or superoxide/hydrogen peroxide (from NOX, following phorbol 12-myristate 13-acetate (PMA), ATP analogue (BzATP), interleukin-1β (IL-1β) or arachidonic acid (AA) addition) on neuronal survival. RESULTS: Induction of glial iNOS caused little neuronal death. Similarly, activation of NOX alone resulted in little or no neuronal death. However, if NOX was activated (by PMA or BzATP) in the presence of iNOS (induced by LPS and interferon-γ) then substantial delayed neuronal death occurred over 48 hours, which was prevented by inhibitors of iNOS (1400W), NOX (apocynin) or a peroxynitrite decomposer (FeTPPS). Neurons and glia were also found to stain positive for nitrotyrosine (a putative marker of peroxynitrite) only when both iNOS and NOX were simultaneously active. If NOX was activated by weak stimulators (IL-1β, AA or the fibrillogenic prion peptide PrP106-126) in the presence of iNOS, it caused microglial proliferation and delayed neurodegeneration over 6 days, which was prevented by iNOS or NOX inhibitors, a peroxynitrite decomposer or a NMDA-receptor antagonist (MK-801). CONCLUSION: These results suggest a dual-key mechanism, whereby glial iNOS or microglial NOX activation alone is relatively benign, but if activated simultaneously are synergistic in killing neurons, through generating peroxynitrite. This mechanism may mediate inflammatory neurodegeneration in response to cytokines, bacteria, ATP, arachidonate and pathological prions, in which case neurons may be protected by iNOS or NOX inhibitors, or scavengers of NO, superoxide or peroxynitrite.