Different pathways for iNOS-mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression.
Co-localization of activated microglia and damaged neurones seen in brain injury suggests microglia-induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co-cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)-derived NO aloneis responsible for neuronal death from interferon gamma (IFNgamma) +lipopolysaccharide (LPS)-activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO-mediated toxicity to immature neurones, MK801 rescues 60-70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO-donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO-mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.
Cambridge Centre for Brain Repair, University of Cambridge, Cambridge, UK. sg274@cus.cam.ac.uk
Sabine
S
Golde
Siddharthan
S
Chandran
Guy C
GC
Brown
Alastair
A
Compston
D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D017628:Microglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018696:Neuroprotective Agents / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis / Q000633:toxicity; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D015398:Signal Transduction / Q000502:physiology*; D005680:gamma-Aminobutyric Acid / Q000493:pharmacokinetics
D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D008070:Lipopolysaccharides; D018696:Neuroprotective Agents; D020030:Nitric Oxide Donors; D016194:Receptors, N-Methyl-D-Aspartate; D009569:Nitric Oxide; D005680:gamma-Aminobutyric Acid; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat